Methods for the simultaneous detection of HCV antigens and HCV antibodies

ABSTRACT

The subject invention relates to methods for the simultaneous detection of Hepatitis C Virus (HCV) antigens as well as antibodies produced in response to HCV antigens. Furthermore, the subject invention allows one to detect antigens in the early, acute stage of infection, even prior to the development of antibodies, thereby allowing for early detection of infected blood and blood products, thus improving the safety of the blood supply.

[0001] The subject application is a Continuation-In-Part of pending U.S.patent application Ser. No. 09/891,983, filed on Jun. 26, 2001, herebyincorporated in its entirety by reference.

BACKGROUND OF THE INVENTION

[0002] 1. Technical Field

[0003] The subject invention relates to methods for the simultaneousdetection of Hepatitis C Virus (HCV) antigens as well as antibodiesproduced in response to HCV antigens. Furthermore, the subject inventionallows one to detect antigens in the early, acute stage of infection,even prior to the development of antibodies, thereby allowing for earlydetection of infected blood and blood products, and thus improving thesafety of the blood supply.

[0004] 2. Background Information

[0005] Recent epidemiological studies indicate that HCV infects morethan 170 million people worldwide and that, in more than 50% of thecases, the infection is chronic. In the United States, there areapproximately 4 million people infected, and 30,000 new infections areestimated to occur annually (NIH Conference, Hepatology Suppl 1:2S(1997)). In addition, HCV is responsible for 8,000-10,000 deathsannually in the United States and is the leading indicator for livertransplantation.

[0006] The HCV genome is a single-stranded RNA molecule of positivepolarity that is approximately 9400-9500 nucleotides in length. Theorganization of the coding regions resembles that of other flaviviruses[Major et al., Hepatology 25:1527 (1997)] as well as the more recentlydiscovered GB viruses [Muerhoff A S, et al., J Virol 69:5621 (1995)].The HCV genome possesses a large open reading frame (ORF) encoding apolyprotein precursor of 3010 to 3033 amino acids depending on theparticular isolate [Choo et al., Proc Natl Acad Sci USA 88:2451 (1991);Grakoui et al., J Virol 67:1385 (1993)]. HCV structural genes (core andenvelope) are encoded near the 5′-end of the genome, followed by theproteases and helicase, the helicase cofactor and the replicase.Noncoding regions (NCR), thought to be important in replication, arefound at each end of the genome.

[0007] HCV infection occurs primarily through parenteral exposure, i.e.,through shared needles, by tattooing, or through transfusion ofcontaminated blood or blood products. Following exposure, the virusenters a susceptible hepatocyte and viral replication occurs. There isan eclipse phase period of approximately 10 days during which time thereis no evidence of viral presence (i.e., viral RNA cannot be detected),serum transaminase levels are within normal limits, and there is noevidence of an immune response to HCV [Busch et al., Transfusion 40:143(2000)]. Typically, about 10 days following exposure, HCV RNA can bedetected, often with viral loads between 100,000-120,000,000 HCV RNAcopies per ml of serum. Several weeks later, there is typically anincrease in ALT levels indicating inflammation of the liver; antibodiesare detected an average of about 70 days after exposure.

[0008] One of the preventive measures employed to limit the spread ofHCV infections is to screen blood for exposure to HCV, either by thedetection of antibodies to HCV or by the detection of viral-specificmolecules (e.g., HCV RNA or HCV core proteins) in serum/plasma. Blood orblood products derived from individuals identified as having beenexposed to HCV, by these tests, are removed from the blood supply andare not utilized for distribution to recipients of blood products (see,e.g., U.S. Pat. No. 6,172,189). These tests may also be utilized in theclinical setting to diagnose liver disease attributable to HCVinfection.

[0009] Due to the unavailability of native, intact HCV virions,serologic antibody tests have relied on recombinant antigens orsynthetic peptides, representing selected fragments of the viralpolyprotein. The first generation anti-HCV screening tests were based ondetection of antibodies directed against a recombinant protein (HCVgenotype 1a) originating from sequences located in the nonstructuralNS-4 protein (C100-3) [Choo et al., Science 244:359 (1989); Kuo et al.,Science 244:362 (1989)]. The first generation assays failed to detectantibodies in approximately 10% of individuals having chronic HCVinfection and up to 10-30% of individuals presenting with acute HCVinfection. The second generation anti-HCV assays have incorporatedrecombinant proteins from three different regions of the HCV genome (HCVgenotype 1a), including amino acid sequences from the core, NS3, and NS4protein [Mimms et al., Lancet 336:1590 (1990); Bresters et al., Vox Sang62:213 (1992)], allowing a marked improvement over the first generationtests in identifying HCV infected blood donors [Aach et al., N Engl JMed 325:1325 (1991); Kleinman et al., Transfusion 32:805 (1992)]. Thesecond generation assays detect antibodies in close to 100% of chronicHCV cases [Hino K., Intervirology 37:77 (1994)] and in nearly 100% ofthe acute cases by 12 weeks post infection [Alter et al., N Engl J Med327:1899 (1992); Bresters et al., Vox Sang 62:213 (1992)]. The thirdgeneration test includes a recombinant protein expressing amino acidsequences from the NS5 region, as well as antigens from the core, NS3and NS4. Some studies have indicated a slight improvement in sensitivityin comparing the third generation tests to second generation tests [Leeet al., Transfusion 35:845 (1995); Courouce et al. Transfusion34:790-795 (1994)], but this improvement is largely attributed tochanges in the NS3 protein rather than the inclusion of NS5 [Courouce etal., Lancet 343:853 (1994)].

[0010] In general, the second and third generation HCV antibody testsdetect exposure to HCV about 70 days after exposure. Since HCVestablishes persistent, and in many cases lifelong infection, thedetection of antibodies to HCV represents a very efficient method fordetermining exposure to HCV. However, antibody testing alone willfrequently fail to detect HCV infected individuals during the first 70days after exposure.

[0011] The existing HCV antigen tests rely on detecting the presence ofthe HCV core antigen in serum or plasma. The core (or nucleocapsid)protein comprises the first 191 amino acids of the polyprotein. Twodifferent types of serologic assays have been developed which permitdetection of HCV core antigens in serum. One assay format detects HCVcore antigens in subjects prior to seroconversion and is utilized inscreening blood donors, while the other assay format detects coreantigens only in hepatitis C patients, regardless of their HCV antibodystatus and is utilized in clinical laboratories to diagnose exposure toHCV or to monitor antiviral therapy.

[0012] Recent data on samples obtained during the pre-seroconversionperiod indicate that the HCV antigen test detects exposure to HCVsignificantly earlier than antibody testing [Aoyagi et al., J ClinMicrobiol 37:1802 (1999); Peterson et al., Vox Sang 78:80(2000); Dawsonet al., Transfusion, SD161, 40(2000); Muerhoff et al., 7^(th)International Meeting on Hepatitis C virus and related viruses, Dec.3-7, 2000], and represents an alternative to nucleic acid testing fordetecting exposure to HCV during the pre-seroconversion period. Theadvantages of HCV antigen detection are that the test is rapid, simple,may-not require sample extraction or other pretreatment, and is not asprone to handling errors (e.g., contamination) as may occur in the HCVRNA tests.

[0013] In clinical laboratories, the HCV antigen test has comparablesensitivity to the HCV DNA tests in detecting exposure to HCV inpatients infected with different HCV genotypes [Dickson et al.,Transplantation 68:1512 (1999)] and in monitoring antiviral therapy[Tanaka et al., Hepatology 32:388 (2000); Tanaka et al., J Hepatol23:742 (1995)]. Thus, HCV core antigen tests present a practicalalternative to HCV RNA for screening blood donors or for monitoringantiviral therapy.

[0014] The uniqueness of the current invention lies in its ability todetect HCV antibodies and HCV antigens simultaneously (see alsoInternational Application No. PCT/JP99/04129). This combination test or“combo” assay utilizes antigen detection to identify exposure to HCVduring the pre-seroconversion “window period” and antibody detection toidentify exposure to HCV after seroconversion.

[0015] All U.S. patents and publications referred to herein are herebyincorporated in their entirety by reference.

SUMMARY OF THE INVENTION

[0016] The subject invention encompasses a method of simutaneouslydetecting at least one Hepatitis C Virus (HCV) antigen and at least oneHCV antibody in a test sample comprising the steps of: a) contacting thetest sample with: 1) at least one HCV viral antigen or portion thereofcoated on a solid phase (e.g., a microparticle), for a time and underconditions sufficient for the formation of antibody/antigen complexesand 2) at least one antibody to HCV or portion thereof coated on thesolid phase, for a time and under conditions sufficient for theformation of antigen/antibody complexes; b) detecting theantibody/antigen complexes, presence of the complexes indicatingpresence of at least one HCV antigen in the test sample; and c)detecting the antigen/antibody complexes, presence of the complexesindicating presence of at least one HCV antibody in the test sample. Theat least one HCV antigen coated on the solid phase may be, for example,core antigen, NS3, NS4, NS5, and portions (or fragments) thereof. The atleast one antibody coated on the solid phase may be, for example, amonoclonal antibody selected from the group consisting of 13-959-270,14-1269-281, 14-1287-252, 14-153-234, 14-153-462, 14-1705-225,14-1708-269, 14-1708-403, 14-178-125, 14-188-104, 14-283-112,14-635-225, 14-726-217, 14-886-216, 14-947-104, 14-945-218, 107-35-54,110-81-17, 13-975-157, 14-1350-210, C11-3, C11-7, C11-10, C11-14 andC11-15. Further, the at least one monoclonal antibody coated on thesolid phase preferably is not reactive with the at least one antigencoated on the solid phase. In particular, the at least one monoclonalantibody may be a HCV anti-core monoclonal antibody and the at least oneantigen may be a recombinant HCV core protein. The recombinant coreprotein does not contain epitopes to which the anti-core monoclonalantibody binds.

[0017] Additionally, the present invention includes a method forsimultaneously detecting the presence of at least one HCV antigen and atleast one HCV antibody in a test sample comprising the steps of: a)contacting the test sample with: 1) at least one HCV viral antigen orportion thereof coated on a solid phase, wherein the solid phase is, forexample, a microparticle, for a time and under conditions sufficient forthe formation of antibody/antigen complexes and 2) at least one HCVantibody or portion thereof coated on the solid phase, for a time andunder conditions sufficient for the formation of antigen/antibodycomplexes; b) adding a first conjugate to the resulting antibody/antigencomplexes for a time and under conditions sufficient to allow theconjugate to bind to the bound antibody in (a) (1), wherein theconjugate comprises a second antibody (e.g., mouse anti-human IgG)attached to a label (for example, a chemiluminescent compound) capableof generating a detectable signal and simultaneously adding a secondconjugate to the resulting antigen/antibody complexes for a time andunder conditions sufficient to allow said second conjugate to bind tothe bound antigen in (a) (2), wherein said second conjugate comprises athird antibody (e.g., a monoclonal antibody to anti —HCV core antigensuch as C11-10) attached to the label, for example, chemiluminescentcompound, capable of generating a detectable signal; and b) detectingthe presence of the generated signal, presence of the signal indicatingthe presence of at least one HCV antigen or at least one HCV antigen inthe test sample. Again, the at least one HCV antigen coated on the solidphase may be selected from the group consisting of core antigen, NS3,NS4, NS5, and portions thereof. Further, the at least one antibodycoated on the solid phase may be a monoclonal antibody selected from thegroup consisting of, for example, 13-959-270, 14-1269-281, 14-1287-252,14-153-234, 14-153-462, 14-1705-225, 14-1708-269, 14-1708-403,14-178-125, 14-188-104, 14-283-112, 14-635-225, 14-726-217, 14-886-216,14-947-104, 14-945-218, 13-975-157, 14-1350-210, 107-35-54, 110-81-17,C11-3, C11-7, C11-10, C11-14 and C11-15.

[0018] The at least one monoclonal antibody coated on the solid phase ispreferably not reactive with the at least one antigen coated on thesolid phase.

[0019] Also, the present invention encompasses a kit comprising: a) acontainer containing at least one HCV antigen coated on a solid phase,wherein the solid phase is, for example, a microparticle; and b) acontainer containing at least one HCV antibody coated on a solid phase,wherein the solid phase is preferably a microparticle.

[0020] The present invention also includes a kit comprising: a containercontaining: 1) at least one HCV antigen coated on a solid phase, whereinthe solid phase is preferably a microparticle, and 2) at least one HCVantibody, coated on the solid phase. The kit may further comprise atleast one conjugate comprising a signal-generating compound attached toa HCV antigen or HCV antibody. The signal-generating compound may be,for example, acridinium or an acridinium-containing compound.

[0021] Additionally, the present invention includes a method ofdetecting HCV antigen in a test sample comprising the steps of: a)contacting the test sample with at least one HCV antibody (e.g.,monoclonal) coated on a solid phase, wherein the solid phase is amicroparticle, for a time and under conditions sufficient for theformation of antibody/antigen complexes; and b) detecting the presenceof antibody/antigen complexes, presence of the complexes indicatingpresence of antigen in the test sample.

[0022] The invention also encompasses a method of detecting HCV antigenin a test sample comprising the steps of: a) contacting the test samplewith at least one HCV antibody (e.g., monoclonal) coated on a solidphase, wherein the solid phase is, preferably, a microparticle, for atime and under conditions sufficient for the formation ofantibody/antigen complexes; b) adding a conjugate to the resultingantibody/antigen complexes for a time and under conditions sufficient toallow the conjugate to bind to the bound at least one antibody, whereinthe conjugate comprises a second antibody attached to a label, forexample, a chemiluminescent compound capable of generating a detectablesignal; and c) detecting the signal generated by the label, for example,chemiluminescent compound, a signal generated by the label indicatingthe presence of antigen in the test sample.

[0023] Also, the present invention includes a recombinant proteincomprising an amino acid sequence selected from the group consisting of,for example, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:12 and SEQ ID NO:16 aswell as an amino acid sequence comprising conservative amino acidsubstitutions of these sequences. (A conservative substitution isdefined as one or more amino acid substitutions in a sequence which donot change the function of the sequence.) The present invention alsoincludes a recombinant protein comprising an amino acid sequence encodedby a nucleotide sequence selected from the group consisting of, forexample, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:11 and SEQ ID NO:15.

[0024] (Substitutions, deletions and additions within the sequenceswhich do not affect functionally affect the protein encoded by thesequence are also considered to be within the scope of the presentinvention.)

[0025] Additionally, the present invention includes a vector orconstruct comprising a nucleotide sequence selected from the groupconsisting of, for example, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:11 andSEQ ID NO:15. The invention also includes a host cell comprising thevector or construct.

[0026] Furthermore, the present invention includes an immunoassay whichmay simultaneously detect at least one HCV antigen or at least one HCVantibody in a test sample.

BRIEF DESCRIPTION OF THE DRAWINGS

[0027]FIG. 1 illustrates the Abbott PRISM® HCV Ab assay format. Theassay uses a 2-step format that consists of microparticles coated withrecombinant HCV antigens from the core, NS3, NS4 and NS5 regions of theHCV genome. These microparticles, when combined with the donor specimen,a diluent, and a complex of goat polyclonal anti-human F(ab′)₂fragment/murine monoclonal anti-biotin:Acridinium conjugate yield anamount of photons representing a qualitative measurement of anti-HCVantibodies in the specimen when triggered with the PRISM® Activatorsolution.

[0028]FIG. 2 illustrates the HCV Ag assay format. This assay also uses a2-step format. When microparticles coated with HCV Mab (e.g., c11-14)are combined with the donor specimen, a diluent and acridinium-labeledMab (e.g., acridinium labeled c11-10), an amount of photons representinga qualitative measurement of anti-HCV antigens in the specimen willresult. The measured amount of photons indicates the amount of HCVantigens in the specimen when triggered with the PRISM® Activatorsolution.

[0029]FIG. 3 illustrates the HCV Ag/Ab combo assay format. The assayuses a 2-step format. When HCV recombinant antigen and monoclonalantibody blended microparticles (e.g., HCV peptide from the core, andrecombinant antigens from the NS3, NS4 and NS5 regions of the HCV genomeblended with microparticles coated with c11-14) are combined with thedonor specimen, a diluent and blended Acridinium-labeled Mabs (e.g.,acridinium labeled c11-10 and acridinylated mouse-anti-human IgG), anamount of photons representing a qualitative measurement of anti-HCVantigens or anti-HCV antibodies or both in the specimen will result whentriggered with PRISM® Activator solution.

[0030]FIG. 4 lists all of the nucleotide and amino acid sequencesreferred to herein as well as the corresponding sequence identifiernumbers.

DETAILED DESCRIPTION OF THE INVENTION

[0031] The subject invention relates to various methods which may beutilized in order to simultaneously detect antigens of HCV andantibodies to HCV in a biological sample. Thus, if an individual haseither developed specific antibodies to HCV and/or has HCV specificantigens in the biological sample tested, the methods of the presentinvention will yield a positive result. Such results may be used, forexample, to diagnose the patient in terms of presence and status ofinfection (i.e., acute or chronic) as well as to determine thesuitability of a donor blood or blood product sample for transfusion.

[0032] Also, the present invention overcomes the problems associatedwith the “window period” (i.e., 50-60 days post infection) wherein anindividual may be infected with HCV but may not have developedantibodies yet. Such individuals may transmit HCV to others during thisperiod. Thus, by detecting HCV during this “window period”, the presentinvention allows for a quick diagnosis of HCV, as opposed to waiting forthe development of antibodies, and prevents contamination of the bloodsupply.

[0033] In one embodiment of the present invention, HCV viral antigens(e.g., core, N3, N4 and N5), or portions thereof, are coated on a solidphase (or are in a liquid phase). The test or biological sample (e.g.,serum, plasma, urine, etc.) is then contacted with the solid phase. Ifantibodies are present in the sample, such antibodies bind to theantigens on the solid phase and are then detected by either a direct orindirect method. The direct method comprises simply detecting presenceof the complex itself and thus presence of the antibodies. In theindirect method, a conjugate is added to the bound antibody. Theconjugate comprises a second antibody, which binds to the first boundantibody, attached to a signal-generating compound or label. Should thesecond antibody bind to a bound first antibody, the signal-generatingcompound generates a measurable signal. Such signal then indicatespresence of the first antibody in the test sample.

[0034] Examples of solid phases used in diagnostic immunoassays areporous and non-porous materials, latex particles, magnetic particles,microparticles (see U.S. Pat. No. 5,705,330), beads, membranes,microtiter wells and plastic tubes. The choice of solid phase materialand method of labeling the antigen or antibody present in the conjugate,if desired, are determined based upon desired assay format performancecharacteristics.

[0035] As noted above, the conjugate (or indicator reagent) willcomprise an antibody (or perhaps anti-antibody, depending upon theassay), attached to a signal-generating compound or label. Thissignal-generating compound or “label” is itself detectable or may bereacted with one or more additional compounds to generate a detectableproduct. Examples of signal-generating compounds include chromogens,radioisotopes (e.g., 125I, 131I, 32P, 3H, 35S and 14C), chemiluminescentcompounds (e.g., acridinium), particles (visible or fluorescent),nucleic acids, complexing agents, or catalysts such as enzymes (e.g.,alkaline phosphatase, acid phosphatase, horseradish peroxidase,beta-galactosidase and ribonuclease). In the case of enzyme use (e.g.,alkaline phosphatase or horseradish peroxidase), addition of a chromo-,fluro-, or lumo-genic substrate results in generation of a detectablesignal. Other detection systems such as time-resolved fluorescence,internal-reflection fluorescence, amplification (e.g., polymerase chainreaction) and Raman spectroscopy are also useful.

[0036] Examples of biological fluids which may be tested by the aboveimmunoassays include plasma, urine, whole blood, dried whole blood,serum, cerebrospinal fluid, saliva, tears, nasal washes or aqueousextracts of tissues and cells.

[0037] At the same time as the antibodies are being detected, HCVantigens are also being detected; thus, the present invention obviatesthe need for the running of two different tests. This is accomplished byexposing the test sample to a solid phase (or liquid phase) coated withspecific antibodies to HCV (e.g., human or animal monoclonal antibodiesto core, polyclonal antibodies, chimeric antibodies, etc.). Antigens, ifpresent in the sample, bind to the solid phase and may then be detectedby a direct or indirect method as described above. More specifically,the indirect method involves the addition of a conjugate comprising asecond antibody (which binds to the bound antigen) attached to a labelor signal-generating compound. When the second antibody binds to thebound antigen, a detectable signal is then generated indicating presenceof HCV antigen in the test sample.

[0038] The antibodies which are coated on the solid phase as well as the“second antibody” may be, as noted above, monoclonal antibodies orpolyclonal antibodies. For example, if one chooses to utilize monoclonalantibodies, they may be selected from Abbott monoclonal antibodies13-959-270, 14-1269-281, 14-1287-252, 14-153-234, 14-153-462,14-1705-225, 14-1708-269, 14-1708-403, 14-178-125, 14-188-104,14-283-112, 14-635-225, 14-726-217, 14-886-216, 14-947-104 and14-945-218. The following anti-core monoclonal antibodies may also beutilized for purposes of the present invention: 107-35-54, 110-81-17,13-975-157, 14-1350-210 (see U.S. Pat. No. 5,753,430) and Tonen HCV coremonoclonals C11-3, 7, 10, 14 and 15 (see PCT Application WO 099/06836),all of which are available from the American Type Culture Collection,10801 University Boulevard, Manassas, Va. 20110-2209. (For a discussionof the manner in which monoclonal antibodies may be created, see Kohlerand Milstein, Nature (1975) 256:494, and reviewed in MonoclonalHybridoma Antibodies: Techniques and Applications, ed. Hurrell (CRCPress, Inc., 1982); see also J. W. Goding in Monoclonal Antibodies:Principles and Practice (Academic Press, N.Y., 1983; see also U.S. Pat.No. 5,753,430).

[0039] It should be noted that HCV core protein may be one possibletarget of the HCV antigen portion of the assay. More specifically, thedetection of the core protein is accomplished by using monoclonalantibodies directed towards epitopes within the core protein. Theseanti-core monoclonals are placed on the solid phase and facilitate thecapture of core antigen proteins from the test sample. For detection ofHCV antibodies in the test sample, recombinant HCV core protein is alsoplaced on the solid phase. It should be noted however that there aresignificant problems associated with the use of a single protein as thetarget for an antigen test and as the capture reagent for antibodydetection, namely there is significant “cross-reactivity” between thecore antigen and the anti-core monoclonal antibodies coated onto thesolid phase(s). This results in a false positive signal, even in theabsence of the test sample, since the monoclonal antibodies will bind toepitopes present on the recombinant protein.

[0040] In order to avoid such cross-reactivity, the core protein used inthe antibody detection portion of the assay may be modified such thatthe ability of the anti-core monoclonals to bind HCV core is eliminated.Such modification may be achieved by use of recombinant DNA technologyin which the epitope region (i.e., the short sequence of amino acidsneeded for monoclonal antibody binding) is eliminated or modified. Thus,use of the modified recombinant core protein would consequently maintainseveral human epitopes to which antibodies present in the serum ofinfected individuals would bind; however, the anti-core monoclonalantibodies used for antigen capture would not bind the modified protein.Alternatively, one could replace the HCV core recombinant protein withpolypeptides that include sequences known to bind to antibodies presentin the serum of most infected individuals, but do not include sequencescontaining the epitopes recognized by the anti-core monoclonals used todetect HCV core antigens.

[0041] More specifically, as noted above, in order to avoidcross-reactivity, one may use core antigens for antibody detection inthe assay. In particular, in the present invention, the solid phase maybe coated with nonstructural proteins (NS) 3, 4 and/or 5 (i.e., NS3, NS4and/or NS5) and/or the core protein. Alternatively, in the presentinvention, the solid phase may be coated with any of the above-mentionedHCV proteins, or segments or portions thereof, either individually or incombination (for antibody detection). The antigens used for coating thesolid phase may be generated as a contiguous recombinant protein,expressed as recombinant proteins, either as a single entity or asdiscrete entities, or as synthetic peptides designed either as a singleentity or discrete entities.

[0042] It should also be noted that one may also detect antibodies toHCV E2 in the combo assay. Thus, using the present assay describedherein, one may replace an assay which detects anti-core antibody.Alternatively, one may supplement such an anti-core antibody assay withthe antigen assay portion of the combo assay described herein. (See,e.g., U.S. Pat. No. 6,156,495 relating to detection of HGBV E2 antibodyor antigen.) With respect to detection of antigens in the presentinvention, as noted above, the monoclonal or polyclonal antibodiescoated on the solid phase must not recognize the core antigens used onthe solid phase (for antibody detection). Thus, for example, in thepresent invention, one may use the full antibody or a fragment thereof.(For purposes of the present invention, a “fragment” or “portion” of anantibody is defined as a subunit of the antibody which reacts in thesame manner, functionally, as the full antibody with respect to bindingproperties.)

[0043] Additionally, it should also be noted that the initial captureantibody (for detecting HCV antigens) used in the immunoassay may becovalently or non-covalently (e.g., ionic, hydrophobic, etc.) attachedto the solid phase. Linking agents for covalent attachment are known inthe art and may be part of the solid phase or derivatized to it prior tocoating.

[0044] The second manner in which to use the solid phase for detectingHCV antibodies involves elimination of the core antigens entirely. Forexample, the solid phase is coated with NS3, NS4 and/or NS5 and asubstitute for the core protein or regions thereof (e.g., E2). Incontrast, the antibodies coated on the solid phase for detection ofantigen are directed against the core protein of HCV.

[0045] Other assay formats which may be used for purposes of the presentinvention, in order to simultaneously detect antigens and antibodiesinclude, for example, Dual assay strip blots, a rapid test, a Westernblot, as well as the use of paramagnetic particles in, for example, anArchitect® assay (Frank Quinn, The Immunoassay Handbook, Second edition,edited by David Wild, pages 363-367, 2001). Such formats are known tothose of ordinary skill in the art.

[0046] It should also be noted that the assays of the present inventionmay also be used to solely detect HCV antigens or HCV antibodies, ratherthan both, if desired. Certainly, if one desires to establish that aninfection initially exists, one may simply want to determine thepresence of antigen in a test sample such as during the “window period”.On the other hand, if one wants to establish the stage of infection(e.g., acute versus chronic), one may wish to look for the presence ofantibodies and titer thereof.

[0047] It should also be noted that the elements of the assay describedabove are particularly suitable for use in the form of a kit. The kitmay also comprise one container such as vial, bottles or strip, witheach container with a pre-set solid phase, and other containerscontaining the respective conjugates. These kits may also contain vialsor containers of other reagents needed for performing the assay, such aswashing, processing and indicator reagents.

[0048] The present invention may be illustrated by the use of thefollowing non-limiting examples:

EXAMPLE I Mapping of HCV Core Epitopes Recognized by MonoclonalAntibodies

[0049] To determine the region within the HCV core protein to which eachof the monoclonal antibodies binds, a series of overlapping,biotinylated peptides were synthesized (Table I). These peptides wereused to develop EIAs, as described below. It should be noted that allmonoclonals were able to detect a recombinant HCV core fusion proteinusing EIA methodologies similar to that described below (data notshown). TABLE I HCV-Core Derived Peptides Core Region Peptide SequenceRepresented A MSTNPKPQKKNKRNTNRR  1-18 B NKRNTNRRPQDVKFPGGG 11-28 CDVKFPGGGQIVGGVYLLP 21-38 D VGGVYLLPRRGPRLGVRA 31-48 E GPRLGVRATRKTSERSQP41-58 F KTSERSQPRGRRQPIPKA 51-68 G RRQPIPKARRPEGRTWAQ 61-78 HPEGRTWAQPGYPWPLYGN 71-88 I QYPWPLYGNEGCGWAGWLL 81-98 J CGWAGWLLSPRGSRPSW 91-107 1 WLLSPRGSRPSWGPTDPRRRSRNLG  96-120 2 SWGPTDPRRRSRNLGKVIDTLTCGF106-130 3 SRNLGKVIDTLTCGFADLMGYIPLV 116-140 4 LTCGFADLMGYIPLVGAPLGGAARA126-150 5 YIPLVGAPLGGAARALAH GVRVLED 136-160 6 GAARALAHGVRVLEDGVNYATGNLP146-170 7 LEDGVNYATGNLPGCSFSIFLLA 158-180 8 LPGCSFSIFLLALLSCLTVPASA169-191

[0050] Coating of polystyrene beads: One quarter-inch polystyrene beadswere used as the solid phase for the peptide EIAs. Prior to coating,beads were washed with 15% isopropanol (in water) at room temperaturefor 30 minutes without agitation. Isopropanol was removed and the beadswere rinsed once with deionized water. The washed beads were then addedto a vial containing the peptide diluted to 5 μg/ml in 0.1 M sodiumphosphate, pH 7.5 buffer (0.233 ml per bead). Beads were incubated at56° C. for 2 hours with end-over-end mixing. Beads were then washedthree times with PBS and then incubated in PBS containing 0.1% TritonX-100 at 40° C. for 1 hour with end-over-end mixing. They were againwashed three times in PBS and then incubated at 40° C. in 5% BSA/PBS for1 hour end-over-end mixing. Beads were washed four times with PBS andthen incubated at room temperature in PBS containing 5% sucrose withoutmixing for 20 minutes. Sucrose buffer was removed and beads air-dried.Coated beads were stored desiccated at 4° C.

[0051] Bead coating validation: To determine whether the biotinylatedpeptides were actually coated onto the beads, an assay was performed inwhich beads were incubated in buffer containing horseradishperoxidase-labeled streptavidin (200-400 ng/ml). The beads were thenwashed with deionized water and substrate added. Product was detected byabsorbance at 492 nm. All peptides in Table I were shown to be coatedonto the polystyrene beads by this assay (data not shown).

[0052] HCV peptide EIAs: Monoclonal antibodies generated against arecombinant HCV core protein (see Example I) were tested for theirability to bind to each of peptide-coated beads as follows: monoclonalsantibodies were diluted to 50 ng/ml in sample diluent buffer (Trisbuffer containing 20% goat serum, 10% calf serum, 0.2% Triton X-100 andsodium azide) of which 0.2 ml was added into a reaction well containingthe peptide-coated bead and incubated at room temperature for 2 hourswith mixing. Beads were then washed with deionized water followed by theaddition of 0.2 ml of peroxidase-labeled goat anti-mouse IgG (0.3μg/ml). Beads were incubated at room temperature for 60 minutes withmixing. Beads were washed with deionized water, transferred into plastictubes to which 0.3 ml of OPD (0.3% O-phenylenediamine-2-HCl in citratebuffer containing 0.02% H₂O₂) substrate was added, and incubated in thedark at room temperature for 30 min without mixing. Reaction wasquenched by the addition of 1 ml of 1N H2SO4 and the OD at 492 nmdetermined. The absorbance is directly proportional to the amount ofantibody bound to the bead.

[0053] Peptide mapping of monoclonals: Using the assay as describedabove, each of the monoclonals were assayed for their ability to bindeach of the HCV-core-derived peptide coated beads. When a monoclonalantibody was found to bind to a specific peptide-coated bead, 10-foldserial dilutions of the monoclonal antibody were made which were thenassayed for binding to the same peptide. This allowed the determinationof binding specificity for each monoclonal antibody. Results shown inTable II indicate the lowest dilution of monoclonal antibody thatexhibited binding (absorbance at least 3-times background). TABLE IIAnti-core monoclonal peptide mapping Peptide A B C D E F G H IMonoclonal aa1-18 aa11-28 aa21-37 aa31-48 aa41-57 aa51-68 aa61-78aa71-88 aa81-98 14-1350-210 A07 — — — — — — — — — 13-975-157 A08 — — — —— — — — — 13-959-270 A09 — — — — — — — — — 110-81-17 A15 — — — — — — — —— 107-35-54 A04 — — — — — — — — — 14-1708-269 A269 — — — — —  5 ng/ml —— — 14-1705-255 A10 — — — — — 500 pq/ml — — — 14-1287-252 A12 — — — — — 5 ng/ml — — — 14-1269-281 A03 — — — — —  50 ng/ml — — — 14-947-104 A16— — — — — 500 pq/ml — — — 14-945-218 A218 — — — — — — — — — 14-886-216A14 — — — — — — — — — 14-726-217 A06 — — — — —  50 ng/ml — — —14-635-225 A05 — — — — — — — — — 14-283-112 A112 — — — — —  5 ng/ml — —— 14-188-104 A11 — — — — —  5 ng/ml — — — 14-178-125 A13 — — — — — 500pg/ml — — — 14-153-234 A234 — — — — — 500 pg/ml — — — C11-3 C11-3 — — —— — — — — — C11-7 C11-7 — — — — — — — — — C11-10 C11-10 — — — — — — — —— C11-14 C11-14 — — — — — — — — — C11-15 C11-15 — 50 ng/ml — — — — — — —Peptide 1 2 3 4 5 6 7 8 Monoclonal aa96-120 aa106-130 aa116-140aa126-150 aa136-160 aa146-170 aa158-180 aa169-191 14-1350-210 A07 — — —— — — — — 13-975-157 A08 — 50 ng/ml — — — — — — 13-959-270 A09 — — — — —— — — 110-81-17 A15 50 ng/ml  5 ng/ml — — — — — — 107-35-54 A04 50 ng/ml— — — — — — — 14-1708-269 A269 — — — — — — — — 14-1705-255 A10 — — — — —— — — 14-1287-252 A12 — — — — — — — — 14-1269-281 A03 — — — — — — — —14-947-104 A16 — — — — — — — — 14-945-218 A218 — — — — — — — —14-886-216 A14 — — — — — — — — 14-726-217 A06 — — — — — — — — 14-635-225A05 — — — — — — — — 14-283-112 A112 — — — — — — — — 14-188-104 A11 — — —— — — — — 14-178-125 A13 — — — — — — — — 14-153-234 A234 — — — — — — — —C11-3 C11-3  5 ng/ml — — — — — — — C11-7 C11-7 — 50 pg/ml  5 ng/ml — — —— — C11-10 C11-10 — — — — — — — — C11-14 C11-14 — — — — — — — — C11-15C11-15 — — — — — — — —

EXAMPLE II Epitope Mapping of Monoclonal Antibodies

[0054] A. Preparation of HCV Gene Fragment Library.

[0055] A plasmid containing nucleotides 14-5294 of the H strain of HCV(Ogata et al., Proc. Natl. Acad. Sci. USA 88:3392-3396 (1991)) inpGEM-9Zf(−) (Promega Corp., Madison, Wis.) was partially digested usingDNase I by the following method in order to obtain randomepitope-encoding fragments:

[0056] Five μg aliquots of plasmid DNA were incubated at 15° C. for 10minutes in 0.5 M Tris-HCl, pH 7.6, and 10 mM MnCl₂ in the presence ofanywhere from 0.1 to 0.7 units of DNase I. Aliquots from each digestionwere analyzed by agarose gel electrophoresis. The two digestion mixturescontaining 0.6 and 0.7 units of DNase I were found to give the largestamount of fragments in the 50-200 bp range. These two mixtures werepooled and extracted one time with an equal volume of phenol-chloroform(1:1, v/v) then precipitated by the addition of one tenth volume 3 Msodium acetate and 2.5 volumes 100% ethanol followed by centrifugationat 14,000×g for 10 minutes. The ends of the DNA molecules were then madeblunt using the PCR Polishing kit (Stratagene, Inc., La Jolla, Calif.)as per manufacturer's directions. The DNA was again extracted andprecipitated as described above, followed by ligation to adouble-stranded adaptor in a 10 μl reaction volume using a T4 DNA ligasekit (Stratagene, Inc., La Jolla, Calif.) as directed by themanufacturer. The sequence of this double stranded adaptor was:5′-GATCGCTCGAATTCCTCG-3′ (SEQUENCE ID NO:1) 3′-TTCTAGCGAGCTTAAGGAGC-5′(SEQUENCE ID NO:2)

[0057] The sense-strand oligonucleotide of the adaptor (SEQ ID NO:1) wasthen used as a primer in a PCR reaction such that all DNAs wereamplified independent of their sequence. This method is a modificationof that described by Akowitz et al., Gene 81:295-306 (1989) and Reyes etal., Mol. Cell. Probes 5:473-481 (1991). PCR was performed in thepresence of the sense-strand oligonucleotide primer at a finalconcentration of 1 μM in a reaction volume of 100 μl using the GeneAmpGold PCR kit (PE Applied Biosystems, Foster City, Calif.) as directed bythe manufacturer in a PE-9600 thermocycler. A pre-incubation at 94° C.for 8 min was followed by twenty-five cycles of PCR as follows:denaturation at 94° C. for 20 seconds, annealing at 55° C. for 30seconds, and extension at 72° C. for 1.0 min. This was followed by afinal extension step at 72° C. for 10 min. The PCR product was extractedand precipitated as described above. The entire PCR was run on a 1.2%agarose gel and a gel slice containing DNA fragments betweenapproximately 70 and 250 bp was removed. The DNA was extracted from thegel slice using the QIAEX II kit (QIAGEN, Inc., Valencia, Calif.) as permanufacturer's directions. The DNA was digested with the restrictionenzyme EcoRI (New England Biolabs, Beverly, Mass.) as directed by themanufacturer. The digested DNA was then extracted and precipitated asdescribed above.

[0058] T7Select10-3b (Novagen, Inc., Madison, Wis.) was digested withEcoRI and dephosphorylated with calf intestinal alkaline phosphatase(New England Biolabs, Beverly, Mass.) as directed by the manufacturer.Size-selected digested DNA fragments (30 ng) (supra) were ligated with0.5 μg digested T7Select10-3b in a 5 μl reaction volume at 16° C.overnight. The entire ligate was packaged into phage heads usingT7Select packaging extract (Novagen, Inc., Madison, Wis.) and titered asdirected by the manufacturer. The resulting unamplified librarycontained a total of 3.9×10⁶ members (PFU). The packaged phage wereamplified by liquid lysate amplification in E. coli BLT5403 (20 mlculture) as directed by the T7 Select System Manual (Novagen, Inc.,Madison, Wis.). The amplified library had a titer of 1.3×10¹¹ PFU perml.

[0059] B. Biopanning of HCV Gene Fragment Library.

[0060] Each monoclonal antibody (20 μg) that was to be used forbiopanning was incubated at 4° C. for 4 hours on an end-over-end rockerin 300 μl blocking buffer (2% BSA, 3% nonfat dry milk, 0.2% Tween 20,0.02% sodium azide in phosphate-buffered saline). During the incubationof the monoclonal antibody, an aliquot of the amplified HCV genefragment library (supra) containing approximately 10¹¹ phage wasprecipitated as follows: {fraction (1/10)} volume of 5 M NaCl was addedto the phage, mixed thoroughly, followed by addition of ⅙ volumepolyethylene glycol (MW 8000), mixed thoroughly again, and incubated onice for 1-2 hours. The phage were centrifuged at 6000×g for 10 min atroom temp, all supernatant was removed and the phage pellet wasvigorously resuspended in 120 μl buffer containing 1 M NaCl, 10 mMTris-HCl pH 8.0, 1 mM EDTA. The phage were added to the pre-incubatedmonoclonal antibody and incubated at 4° C. overnight on an end-over-endrocker.

[0061] The next morning, the antibody-phage complexes were captured onparamagnetic particles coupled to goat anti-mouse IgG (Fc specific) asfollows. A 0.2 ml aliquot of Goat Anti-Mouse IgG Fc BioMag particles(Polysciences, Inc., Warrington, PA) was washed three times with 0.4 ml0.1% Tween 20, 0.1% BSA, 0.02% sodium azide in phosphate-buffered saline(PBS) by gentle vortexing followed by capture on a magnetic stand for0.5-1 minute. The supernatant was removed carefully without disturbingthe particles. Particles were then resuspended in the IgG-phage fromovernight incubation above and incubated at room temperature on anend-over-end rocker for 3 hours. Particles were washed six times asabove using 6.0 ml 0.5% Tween 20, 0.1% BSA in PBS per wash. Bound phagewere eluted using 0.2 ml 0.1% Tween 20, 0.1% BSA, 1.0% SDS in PBS atroom temperature on an end-over-end rocker for 90 minutes. The tube wasplaced on a magnetic stand for 1-2 minutes, after which the supernatantcontaining the eluted phage was removed to a clean tube. The samplecontaining eluted phage was titered as directed in the T7 Select SystemManual.

[0062] The eluted phage was amplified as follows. Ten ml LB Broth (GibcoBRL, Gaithersburg, Md.) plus 100 μg/ml ampicillin was inoculated with E.coli BLT5403 and incubated at 37° C. overnight with shaking. Thefollowing morning, 35 ml LB Broth plus 100 μg/ml ampicillin, 1× M9salts, 0.4% glucose, 1 mM MgSO₄ was inoculated with 0.2 ml of theovernight culture and incubated at 37° C. with shaking until the A600absorbance was 0.5-0.6. Eluted phage (185 μl) from first roundbiopanning (supra) was added and incubation at 37° C. was continued for1.5-2 hours, until the A600 absorbance of the culture had dropped toapproximately 0.5, indicating lysis. The culture was centrifuged at8000×g for 10 minutes and the supernatant was removed to a clean tubeand stored at 4° C. The culture supernatant was titered as directed inthe T7 Select System Manual.

[0063] One to two subsequent rounds of biopanning and amplification wereperformed as above with the following modifications. After pre-blockingthe monoclonal antibody for 4 hours at 4° C., 150 μl amplified phagefrom the previous round of biopanning was added instead of 10¹¹PEG-precipitated phage from the starting library. In addition, afterbiopanning, a 20 ml culture rather than a 35 ml culture was used toamplify the eluted phage, and 100 μl rather than 185 μl of eluted phagewas added to the culture.

[0064] C. Selection and Sequencing of HCV Core-Containing Clones.

[0065] A DNA fragment containing a region of the HCV genome that encodesamino acids 1-173 of the HCV nucleocapsid protein was utilized as ahybridization probe. This region was chosen because all of themonoclonal antibodies analyzed in the biopanning experiments recognizeepitopes in the HCV core protein. Phage resulting from 2-3 rounds ofbiopanning and amplification were plated on E. coli BLT5403 andincubated at 37° C. until plaques formed. DNA was transferred ontoHybond —N+membranes (Amersham Life Sciences, Inc., Arlington Heights,Ill.), denatured, neutralized, and UV cross-linked, as described by themanufacturer. The membranes were pre-hybridized, hybridized with the HCVnucleocapsid gene ³²P-labeled probe, washed and exposed as described andknown in the art. Individual hybridizing plaques were isolated and theinserts were amplified by PCR using T7SelectUP and T7SelectDOWN primers(Novagen, Inc., Madison, Wis.) as directed in the T7Select SystemManual. For each monoclonal antibody, 30-50 independent hybridizingplaques were amplified and then purified using the QIAquick PCRpurification kit (Qiagen, Inc., Chatsworth, Calif.). Purified PCRproducts were sequenced directly on an ABI Model 377 DNA Sequencer usingthe ABI Big Dye Terminator Cycle Sequencing Ready Reaction kit(Perkin-Elmer) and the T7SelectUP primer. All of the sequences resultingfrom biopanning with a particular monoclonal antibody were aligned withthe HCV nucleocapsid gene sequence and the minimum region of overlapamong all clones was identified. This overlap region defined the epitoperecognized by the monoclonal antibody. The epitopes recognized byseveral monoclonal antibodies that were identified using this method areshown in TABLE III. TABLE III Monoclonal Region of HCV Antibody CoreRecognized C11-15 Amino acids 19-27 C11-10 Amino acids 32-36 C11-14Amino acids 45-50 C11-3 Amino acids 104-110 C11-7 Amino acids 112-124 14-635-225 Amino acids 49-53  14-153-462 Amino acids 50-63  14-726-217Amino acids 57-63  14-178-125 Amino acids 59-64  14-1269-281 Amino acids59-64  14-947-104 Amino acids 59-64  14-188-104 Amino acids 59-64 14-1708-269 Amino acids 59-64 107-35-54 Amino acids 102-109

EXAMPLE III Construction of Recombinant Antigens for Use in an HCV CoreAntibody/Antigen Combination Assay

[0066] A. Background.

[0067] The human immune response to Hepatitis C Virus (HCV) core is, forthe most part, exclusive to the N-terminal half of the native protein.Multiple epitopes (regions comprising a defined number of amino acids,usually <10) have been identified within the first 115 amino acids ofthe native protein (Sallberg et al). Therefore, recombinant antigensutilized in assays for the detection of human anti-core antibodiespresent in the serum of infected individuals need only contain thisportion of the native protein. Conversely, in vitro assays for thedetection of HCV core protein utilize murine monoclonal antibodies tocapture and detect native core protein also present in the serum ofinfected individuals. Combination assays for the simultaneous detectionof both core antigen and human anti-core antibody in a single assaycombine the two assay formats. In this case, a recombinant core antigenis necessary that will be recognized by human anti-core antibodiespresent in the serum, while escaping recognition by the murinemonoclonal antibodies used to capture and detect native core antigenalso present in serum. Such recombinants can be constructed byeliminating small regions (1-30 or more amino acids) within the coreantigen, thus disrupting or eliminating the epitope(s) recognized by themurine monoclonal antibodies while at the same time leaving undisturbednumerous other epitopes that will allow human anti-core antibodydetection.

[0068] B. Antigen Construction.

[0069] The first of these antigens constructed contained HCV amino acids8-100 in which amino acids 32-50 were deleted from the recombinant. Inthis manner, antibodies C11-10 and C11-14, which bind to epitopes atamino acids 32-36 and 45-50, respectively (Example II), will not bind tothe resulting recombinant antigen. A plasmid containing a bacterialcodon-optimized version of nucleotides 342-791 from the H strain of HCV(Ogata, supra (1991)) was used as template for the PCR. oligonucleotideprimers designed to this sequence were utilized to amplify two distinctfragments (1=SEQ ID NO:1 and SEQ ID NO:2; 2=SEQ ID NO:3 and SEQ ID NO:4)of the HCV core antigen, one upstream of the deletion and the otherdownstream of the deletion. Additionally, the oligonucleotide primersflanking the deletion on each fragment (SEQ ID NO:2 and SEQ ID NO:3)contained regions of overlap with one another. PCR was performed in aPE-9600 thermocycler in the presence of 0.5 μM of each oligonucleotideprimer and 3-5 pg of plasmid template using the TaKaRa LA Taq PCR Kit(Pan Vera, Corp., Madison, Wis.) as per manufacturer's instructions (50μl volume). A pre-incubation at 94° C. for 1 minute was followed by 35cycles of PCR (denaturation at 94° C. for 20 seconds; annealing at 50°C. for 30 seconds; extension at 72° C. for 30 seconds), which was thenfollowed by a final extension at 72° C. for 10 minutes. Following theindependent amplification of the two fragments, each was purified usingthe QIAquick PCR Purification Kit (QIAGEN, Inc., Valencia, Calif.) andeluted into 25 μl of water. A second round of PCR was then performed assupra for 10 cycles to tether the two purified fragments (1 μl each in a20 μl volume reaction) to one another at the regions of overlap withinthe oligonucleotide primers (SEQ ID NO:2 and SEQ ID NO:3). Finally, theproduct of the second PCR was amplified in a third PCR for 35 cyclesutilizing 0.5 μM of the flanking oligonucleotide primers (SEQ ID NO:1and SEQ ID NO:4) and the conditions described supra (100 μl volume).

[0070] C. Recombinant Expression.

[0071] The product of the third PCR was purified using the QIAquick PCRPurification Kit, then digested with the restriction enzymes EcoRI andBamHI, digestion sites that were incorporated into the flankingoligonucleotide primers (SEQ ID NO:1 and SEQ ID NO:4). The fragmentencoding the recombinant was ligated into the bacterial expressionvector pJO200 that had been similarly digested with EcoRI and BamHIusing the pGEM-T Easy Ligation Kit (Promega, Madison, Wis.), thentransformed into XL1-Blue competent cells (Stratagene, La Jolla,Calif.). After selection of clones containing the appropriately sizedinserts, the nucleotide sequence of the recombinant was confirmed (SEQID NO:5), exhibiting the deduced amino acid sequence (SEQ ID NO:6). (SeeU.S. Pat. No. 5,322,769 and U.S. Pat. No. 6,172,189 for a description ofthe expression of recombinant proteins.)

[0072] D. Other Recombinants.

[0073] Several other HCV core recombinants have been constructed, clonedand expressed in a manner identical to that described supra. The firstof these (see nucleotide sequence SEQ ID NO:7 and corresponding aminoacid sequence SEQ ID NO:8) contains HCV amino acids 8-100 in which aminoacids 33-35 and 46-49 have been deleted. The oligonucleotide primersutilized to amplify the two fragments in the first PCR were SEQ ID NO:1and SEQ ID NO:9, and SEQ ID NO:10 and SEQ ID NO:4, respectively,followed by final amplification in the third PCR with SEQ ID NO:1 andSEQ ID NO:4. The second of these recombinants (see nucleotide sequenceSEQ ID NO:11 and corresponding amino acid sequence SEQ ID NO:12) encodesHCV amino acids 8-100 in which the leucine residue at amino acid 36 hasbeen substituted with valine, and the arginine residue at amino acid 47has been substituted with leucine. The oligonucleotide primers utilizedto amplify the two fragments in the first PCR were SEQ ID NO:1 and SEQID NO:13, and SEQ ID NO:14 and SEQ ID NO:4, respectively, followed byfinal amplification in the third PCR with SEQ ID NO:1 and SEQ ID NO:4.Finally, a recombinant (see nucleotide sequence SEQ ID NO:15 andcorresponding amino acid SEQ ID NO:16) encoding HCV amino acids 8-100was constructed using oligonucleotide primers SEQ ID NO:1 and SEQ IDNO:4 in a single PCR reaction.

EXAMPLE IV Construction of Additional Recombinant Antigens for Use in anHCV Core Antibody/Antigen Combination Assay

[0074] A. Background.

[0075] Additional recombinant antigens constructed for use in an HCVantigen/antibody combination assay included antigens that contained the33c region of HCV (amino acids 1192-1457) tethered to a core region ofthe virus. The template used for such constructions was a plasmidcontaining a bacterial codon-optimized sequence of amino acids1192-1457, followed by amino acids 1-150 from the H strain of HCV(Ogata, 1991), with two non-HCV coding amino acids separating the twosequences. This recombinant (HC-43) is routinely used in multiplecommercial assays for the detection of HCV. The HC43 recombinant isexpressed as a non-fusion protein from the pL promoter of bacterialphage lambda. (See U.S. Pat. No. 5,705,330, U.S. Pat. No. 5,616,460 andU.S. Pat. No. 5,773,212 for a discussion of HC43 and U.S. Pat. No.6,153,377 and U.S. Pat. No. 5,859,193 for a discussion of the lambda pLvector system.) The additional recombinants were constructed by one oftwo methods. First, existing clones encoding distinct, relatedrecombinants were joined by DNA ligation to form a third uniquerecombinant, or, new unique clones were constructed by tethering PCRdescribed in Example III, Part B.

[0076] B. Initial Antigen Construction.

[0077] The first of the newer antigens constructed (p9MB-18) containedHCV amino acids 1192-1457 (representing a segment of NS3) tethered toamino acids 1-100 (representing a segment of core protein) in whichamino acids 32-50 had been deleted. This recombinant was constructed byrestricting plasmid pHC43 with the endonucleases, Xma I and Bam HI. XmaI cuts pHC43 near amino acid 24 of the core-encoding region while Bam HIcuts downstream of the translation termination codon. This region ofpHC43 was replaced by DNA ligation using the pGEM-T Easy Ligation Kit(Promega Corp., Madison, Wis.), with the Xma I-Bam HI fragment obtainedfrom the pJO200 vector encoding HCV core amino acids 8-100 with 32-50deleted described in Example III (SEQ ID NO:5 and SEQ ID NO:6). The newplasmid was then used to transform XL1-Blue competent cells (Stratagene,La Jolla, Calif.). After selection of clones containing theappropriately sized insert, the nucleotide sequence of the recombinantwas confirmed (SEQ ID NO:17), exhibiting the deduced amino acid sequencein SEQ ID NO:18.

[0078] C. Other Recombinants Constructed by Tethering PCR.

[0079] Recombinant antigens made by tethering PCR were constructed asdetailed in Example III, Part B. The first of these recombinants,p9MB-19 (SEQ ID NO:19 (nucleotide sequence) and SEQ ID NO:20 (amino acidsequence)), contains HCV amino acids 1192-1457 followed by amino acids8-100 in which amino acids 32-50 had been deleted. The oligonucleotideprimers used to amplify the initial two fragments in the first PCR wereSEQ ID NO:21 and SEQ ID NO:22, and SEQ ID NO:23 and SEQ ID NO:4,respectively. Final amplification in the third PCR utilizedoligonucleotide primers SEQ ID NO:21 and SEQ ID NO:4.

[0080] Recombinant p9MB-20 (SEQ ID NO:24 and SEQ ID NO:25) contains HCVamino acids 1192-1457, four glycine residues and a serine residue,followed by HCV amino acids 8-100 in which amino acids 32-50 had beendeleted. The oligonucleotides primers used to amplify the initial twofragments in the first PCR were SEQ ID NO:21 and SEQ ID NO:22, and SEQID NO:26 and SEQ ID NO:4, respectively. Final amplification in the thirdPCR utilized oligonucleotides primers SEQ ID NO:21 and SEQ ID NO:4.

[0081] Recombinant p9MB-22 (SEQ ID NO:27 and SEQ ID NO:28) contains HCVamino acids 1192-1457, four glycine residues and a serine residue,followed by HCV amino acids 1-150. The oligonucleotides primers used toamplify the initial two fragments in the first PCR were SEQ ID NO:21 andSEQ ID NO:22, and SEQ ID NO:29 and SEQ ID NO:30, respectively. Finalamplification in the third PCR utilized oligonucleotides primers SEQ IDNO:21 and SEQ ID NO:30.

[0082] Recombinant p9MB-31 (SEQ ID NO:31 and SEQ ID NO:32) contains HCVamino acids 1192-1457 followed by amino acids 1-100 in which amino acids31, 32, 33, 47 and 48 had been deleted. The oligonucleotides primersused to amplify the initial two fragments in the first PCR were SEQ IDNO:21 and SEQ ID NO:33, and SEQ ID NO:34 and SEQ ID NO:4, respectively.Final amplification in the third PCR utilized oligonucleotides primersSEQ ID NO:21 and SEQ ID NO:4.

[0083] D. Other Recombinants Constructed by DNA Ligation.

[0084] Recombinant 9MB-24 (SEQ ID NO:35 and SEQ ID NO:36) contains HCVamino acids 1192-1457 followed by amino acids 1-100 in which amino acids33-35 and 46-49 were deleted from the recombinant. This recombinant wasconstructed by restricting plasmid pHC43 with the endonucleases, Xma Iand Bam HI, and replacing the fragment by DNA ligation, with the XmaI-Bam HI fragment obtained from SEQ ID NO:7.

[0085] Recombinant 9MB-25 (SEQ ID NO:37 and SEQ ID NO:38) contains HCVamino acids 1192-1457, four glycine residues and a serine residue,followed by HCV amino acids 1-100 in which amino acids 33-35 and 46-49were deleted from the recombinant. This recombinant was constructed byrestricting plasmid p9MB-22 with the endonucleases, Xma I and Bam HI,and replacing the fragment by DNA ligation, with the Xma I-Bam HIfragment obtained from SEQ ID NO: 7.

[0086] Recombinant 9MB-25 (SEQ ID NO:39 and SEQ ID NO:40) contains HCVamino acids 1192-1457, four glycine residues and a serine residue,followed by HCV amino acids 1-100 in which amino acids 32-50 weredeleted from the recombinant. This recombinant was constructed byrestricting plasmid p9MB-22 with the endonucleases, Xma I and Bam HI,and replacing the fragment by DNA ligation, with the Xma I-Bam HIfragment obtained from p9MB-18 (SEQ ID NO:27).

EXAMPLE V Preparation of Microparticles

[0087] Microparticles, coated with several monoclonal antibodies, wereprepared by coating several separate populations of microparticles withHCV monoclonal antibodies which recognize different regions within theHCV core protein. Similarly, microparticles were coated with recombinantantigens cloned from the NS3 and NS4 regions of HCV. The peptide usedfor microparticle coating was from the core region of HCV.

[0088] Microparticles for Antibody Assay:

[0089] The following recombinant proteins and peptides were used to coatthe microparticles for antibody assays.

[0090] A. Preparation of Recombinant Proteins:

[0091] i. HCV HC43 antigen HCV. HC43 recombinant antigen was obtainedfrom Chiron Corporation, Emeryville, CA. It contained amino acidsequence 1-150 (corresponding to the core protein) and 1192-1457(corresponding to amino acid residues within NS3) of HCV-1 (amino acidsequence available from GenBank®, as described hereinabove)

[0092] ii. HCV C-100 antigen. HCV C-100 recombinant antigen was obtainedfrom Chiron Corporation, Emeryville, Calif. It contained amino acidsequence 1569-1961 (corresponding to amino acid residues within NS4) ofHCV-1 (available from GenBank®, as described hereinabove).

[0093] iii. HCV NS5 antigen. HCV NS5 recombinant antigen was obtainedfrom Chiron Corporation, Emeryville, Calif. It contained amino acidsequence 2054-2995 of HCV (available from GenBank®, describedhereinabove).

[0094] iv. HCV NS3 NS4 E. Coli construct CKS-33c-BCD antigen. HCV HC31recombinant antigen was obtained from Chiron corporation, Emerville,Calif. It contained amino acid Sequence 1192-1457 of HCV, and amino acidsequence 1676-1931 of the NS4 region. In addition, it consists of 239amino acids of CKS (available from GenBank®, described hereinabove).

[0095] A1. Preparation of R-Antigen Coated Microparticles.

[0096] i. Preparation of HCV HC43/C100 Microparticles. Microparticlescoated with both HC43 and c-100 were prepared in the following manner.Briefly, a 500 μl aliquot of microparticles (10% weight/volume, 0.7-0.9micron, available from Seradyn, Indianapolis, Ind.) was mixed with 962μl of a coating buffer (Phosphate buffer, pH 5.0 with Tween-20) forapproximately 1 minute at room temperature. Then, 154 μl of an HCVC100-3 antigen solution (0.65 mg/ml) and 308 ul of an HC43 antigensolution (650 μg/ml) were added to the microparticle solution, mixed andtumbled for 16 hours at room temperature. The microparticles werepelleted at 12,000×g for 10 minutes in an Eppendorf microfuge. Thesuspension was removed, and the microparticles were washed with washbuffer (Phosphate, NaCl, dithiothreitol-DTT, EDTA, sodium dodecylsulfate-SDS, pH 6.5) and heat stressed at 56° C. for 20 hours. Themicroparticles were then resuspended in 2.5 ml of microparticle diluent(Phosphate Buffer, pH 6.5, EDTA, DTT, NaCl and SDS, Sucrose, azide) at afinal concentration of 2.0%.

[0097] ii. Preparation of HCV NS5 Microparticles. Five hundred andthirty microliters of an HCV NS5 coating buffer (Carbonate, pH 10, SDS)and 200 μl of a 10% weight/volume 0.7-0.9 micron microparticles(available from Seradyn, Indianapolis, Ind.) were added to 270 μl of theHCV NS5 antigen solution (concentration of 650 μg/ml). Themicroparticles were mixed and tumbled for 16 hours at room temperature.The microparticles were pelleted at 12,000×g for 10 minutes in anEppendorf microfuge. The suspension was removed and the microparticleswere washed with wash buffer (Phosphate, NaCl, DTT, EDTA, SDS, pH 6.5)and heat stressed at 56° C. for 20 hours. The washed microparticles werethen resuspended in 2.5 ml of microparticle diluent (Phosphate Buffer,pH 6.5, EDTA, DTT, NaCl and SDS, Sucrose, azide) at a finalconcentration of 0.4%.

[0098] iii. Preparation of HCV NS3 NS4 E. Coli Construct CKS-33c-BCDMicroparticles. A 100 μl aliquot of microparticles (10% weight/volume,0.7-0.9 micron, available from Seradyn, Indianapolis, Ind.) was mixedwith 452 μl of coating buffer (Phosphate buffer, pH 5.0 with Tween-20)for approximately 10 minutes at room temperature. Then, 200 μg ofCKS-33C-BCD Ag was added and mixed for 16 hours at room temperature.

[0099] The microparticles were pelleted at 12,000×g for 10 minutes in anEppendorf microfuge. The prepared microparticles were washed with washbuffer (DTT, EDTA, SDS in PBS, pH 6.5). The supernatant was removed, andthe microparticles were resuspended in 1 ml of microparticle diluent(Phosphate Buffer, pH 6.5, EDTA, DTT, NaCl, Sucrose and SDS, Sucrose).

[0100] iv. Blending of HCV HC43/C100 and HCV NS5 Microparticles. Twohundred twenty microliters of HCV HC43/C100 microparticles prepared asdescribed in Example (IV) (A1) (i) and 330 μl of HCV NS5 microparticlesprepared as described in Example (IV) (A1) (ii) were mixed together.This mixture was incubated at room temperature for 15 minutes anddiluted to 50 ml in microparticle diluent. (Phosphate Buffer, pH 6.5,EDTA, DTT, NaCl, Sucrose and SDS, Sucrose).

[0101] v. Preparation of Biotinylated Core Peptide. HCV core peptide aa11-28 was biotinylated at N-terminus during synthesis using an automatedpeptide synthesizer with ≧90% purity.

[0102] vi. Preparation of Streptavidin-Coated Microparticles. A four mlaliquot of carboxylated microparticles (10% weight/volume, 0.227 micron,Seradyn, Indianapolis, Ind) was mixed with 2486 ul of coupling buffer(MES (2-(N-morpholino) ethanesulfonic acid) pH 6.7) for 10 minutes atroom temperature. Then, 114.4 μl of EDAC solution (10 mg/ml in couplingbuffer) was added to the microparticle solution and mixed for 15 minutesat room temperature. Subsequently, 1 ml of Streptavidin solution (1mg/ml in PBS) was added to the activated microparticles and tumbled for16 hours at room temperature. The prepared microparticles were thenpelleted at 12,000×g for 3 minutes in an Eppendorf microfuge. Thesupernatant was removed, and the microparticles were resuspended in 4 mlof PBS. The centrifugation process was repeated one more time, andmicroparticles were stored in 4 ml of PBS to yield a final concentrationof approximately 1%.

[0103] vii. Preparation of Core Peptide Coated Microparticles.

[0104] To 1 ml of coated microparticles from Example (IV) (A1) (vi) wasadded 375 μl of HCV Core peptide from Example (V) (A1) (v) and 11-28 aaat 1 mg/ml in PBS buffer. The mixture was then incubated for 2 hours atroom temperature. The prepared microparticles were washed with washbuffer (DTT, EDTA, SDS in PBS, pH 6.5), and the microparticles wereresuspended in 1 ml of microparticle diluent (Calf Bovine Serum, HorseIgG, TWEEN 20, BSA, Casein, EDTA, Sucrose and Proclin, pH 6.5) yielding1% solids final concentration.

[0105] Microparticles for the Antigen Assay:

[0106] B. Preparation of Monoclonal Antibodies:

[0107] The methods for generating monoclonal antibodies are presented inU.S. Pat. No. 5,753,430. Briefly, E. coli derived recombinant antigensencoded by HCV sequences, designated as pHCV34 (HCV-core, a.a. 1-150),were employed as immunogens for antibodies to core. Detailed informationon the cloning of pHCV34 is disclosed in U.S. patent application Ser.No. 07/572,822, incorporated herein by reference). The protein wasprepared for immunization with appropriate adjuvants after purification,as would be performed by those skilled in the art.

[0108] BALB/c mice were injected intraperitoneally with 15 μg ofpurified pHCV34 with 15 μg each of Trehalose dimycolate (TDM) and M.phlei in a buffer emulsion prepared according to the manufacturer'sinstructions. Subsequent immunizations were performed on day 14, 28 and42. Mice were bled on days 21 and 49, and the immune response wasstudied by enzyme-linked immunosorbent assay utilizing pHCV34 coated onpolystyrene beads, as detailed in U.S. Pat. No. 5,753,430.

[0109] Upon demonstration of specific anti-HCV antibody present atreasonable titers in the sera of immunized mice, mice were boosted with40 μg of pHCV34 antigen. The mice were sacrificed and their spleens wereremoved; the white cells were mixed and fused with SP2/0 cells. The cellmixture was cultured in Biscoe's Modified Dubach's Medium (IMDM)supplemented with 20% fetal calf serum, and the hybridized cells wereselected by using a hypoxanthine and thymidine medium. Hybridoma celllines were established, and all monoclonal antibodies specific forantibodies to core were prepared from ascite fluids of the mice and werepurified by chromatography on a protein-A column (Pharmacia, Uppsala,Sweden). The epitopes of the monoclonal antibodies were analyzed by anELISA test described in Example I.

[0110] B1. Preparation of Monoclonal Antibody-Coated Microparticles forAntigen Assay.

[0111] i. Preparation of HCV C11-14 Microparticles. Briefly, a 1 mlaliquot of carboxylated microparticles (10% weight/volume, 0.227 micron,available from Seradyn, Indianapolis, Ind) was mixed with 9 ml ofcoupling buffer (MES (2-(N-morpholino) ethanesulfonic acid), pH 6.7) forapproximately 10 minutes at room temperature. Then, 150 μl of1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC, 10 mg/ml incoupling buffer, Sigma Chemical Company) was added to the microparticlesolution and mixed for 15 minutes at room temperature. Eighteen hundredand twenty-two microliters of C11-14 monoclonal antibody solution (2.13mg/ml) was added to the activated microparticles, mixed and tumbled for16 hours at room temperature. The microparticles were then pelleted at12,000×g for 3 minutes in an Eppendorf microfuge. The supernatant wasremoved, and the microparticles were washed with microparticle washbuffer (Phosphate Buffer Saline-PBS, Tween 20, pH 7.2), followed bywashing with microparticle coating buffer (Tris Buffer Saline-TBS,Bovine Serum Albumin-BSA, pH 7.2) and final washing with microparticlefinal exchange buffer (PBS, Tween 20, pH 7.2). The microparticles wereresuspended in 5 ml of final exchange buffer and heat stressed at 45° C.for 72 hours. After heat stress, 5 ml of microparticle diluent (CalfBovine Serum, Horse IgG, Tween 20, BSA, Casein, Ethylene diaminetetraacetic acid (EDTA), Sucrose and Proclin, pH 6.5) was added to givea final concentration of approximately 1.0%.

[0112] ii. Preparation of HCV A5 (14-635-225) Mab Microparticles. Asimilar procedure as mentioned in Example (V) (B1) (i) was used, exceptthat instead of C11-14 Mab, A5 14-635-225) Mab was used for coating onmicroparticles.

[0113] iii. Preparation of HCV C11-3 Microparticles. Six point sixmicroliters of 1 N HCl was added to 300 μl (1.45 mg/ml) of C11-3monoclonal antibody to bring the pH to 2.5. The monoclonal was thenincubated at this pH for 5 minutes. The pH was then brought to 6.5 byadding 50 mM MES buffer. A 100 μl aliquot of carboxylated microparticles(10% weight/volume, 0.227 micron, Seradyn, Indianapolis, Ind.) was thenmixed with 333 μl of coupling buffer (MES, pH 6.7) for 10 minutes atroom temperature. Then, 15 μl of EDAC solution (10 mg/ml in couplingbuffer) was added to the microparticle solution and mixed for 5 minutesat room temperature. Five hundred and fifty-two microliters of pHshocked C11-3 monoclonal antibody solution (0.725 mg/ml) were added tothe activated microparticles, mixed and tumbled for 16 hours at roomtemperature. The microparticles were then pelleted at 12,000×g for 3minutes in an Eppendorf microfuge. The supernatant was removed, and themicroparticles were washed with microparticle wash buffer (PhosphatedBuffered Saline (PBS), Tween 20, pH 7.2), followed by a wash withmicroparticle coating buffer (Tris Buffered Saline (TBS), Bovine SerumAlbumin (BSA), pH 7.2) and a final wash with microparticle finalexchange buffer (PBS, Tween 20, pH 7.2). The microparticles wereresuspended in 0.5 ml of final exchange buffer and heat stressed at 45°C. for 72 hours. After heat stress, 0.5 ml of microparticle diluent(Calf Bovine Serum, Horse IgG, Tween 20, BSA, Casein, ethylene diaminetetraacetic acid (EDTA), Sucrose, and ProClin, pH 6.5) was added to givea final concentration of approximately 1.0%.

[0114] iv. Blending of HCV C11-14 and C11-3 Microparticles.

[0115] Thirty-six microliters of HCV C11-3 microparticles (1% solid)prepared as described in Example (V) (B1) (iii) and 84 μl HCV C11-14microparticles (1% solids) prepared as described in Example (V) (B1) (i)were mixed with 880 μl microparticle diluent (Calf Bovine Serum HorseIgG, Tween 20, BSA, Casein, EDTA, Sucrose, and Proclin, pH 6.5).

[0116] C. Preparation of Microparticles for Combo Assay:

[0117] For the dual assay, two separate PRISM® channels were used, onefor the HCV antibody assay and one for the HCV antigen assay. For thecombo assay, both the antibody and antigen assays were performed on asingle channel where the reagents for both antigen and antibody assaywere combined in one kit.

[0118] i. Blending of C11-14 mAb Coated Microparticles with Core Antigen(Peptide)-Coated Microparticles and HCV HC33 Antigen CoatedMicroparticles. Three hundred and fifty microliters of core peptidecoated microparticles (1% solids stock) prepared as in Example (V) (A1)(vii) and 700 μl of HCV NS3 NS4 E. coli Construct CKS-33C-BCD Ag coatedmicroparticles (1% solids stock) prepared as described in Example (V)(A1) (iii) and 319 μl HCV C11-14 microparticles (1.0099% solids stock)prepared as described in Example (V) (B1) (i) were mixed with 5631 μlmicroparticle diluent (calf Bovine Serum, Horse IgG, Tween 20, BSA,Casein, EDTA, Sucrose and Proclin, pH 6.5).

[0119] D. Preparation of Microparticles for Combo Assay Usingp9MB18-Coated Microparticles, c200-Coated Microparticles, andC11-14-Coated Microparticles:

[0120] i. HCV C-200 antigen. HCV C-200 recombinant antigen was obtainedfrom Chiron Corporation, Emeryville, Calif. In particular, the antigencomprises amino acid sequence 1192-1932 of HCV (available from GenBank,as described hereinabove) and is from the NS3 and NS4 regions. The c200antigen is a chimeric fusion protein, with 154 amino acids of humansuperoxide dismutase (hSOD).

[0121] ii. Preparation of c200 Microparticles. A 100 μl aliquot ofmicroparticles (10% % weight/volume, 0.7-0.9 micron, available fromSeradyn, Indianapolis, Ind.) was mixed with 830.6 μl of coating buffer(MES buffer, pH 6.5 with SDS) for approximately 10 minutes at roomtemperature. Then, 69.4 μl of c200 antigen solution (0.72 mg/ml) wasadded to the microparticle solution, mixed and tumbled for 16 hours atroom temperature. The microparticles were pelleted at 12,000×g for 10minutes in an Eppendorf microfuge. The suspension was removed, and themicroparticles were washed with wash buffer (Phosphate, NaCl, EDTA, SDS,pH 6.5) and heat stressed at 56° C. for 16-20 hours. The microparticleswere then resuspended in 500 μl of microparticle diluent (PhosphateBuffer, pH 6.5, EDTA, NaCl, Sucrose, SDS, azide) at a finalconcentration of 2.0%.

[0122] iii. Preparation of p9MB-18 Microparticles. A 100 μl aliquot ofmicroparticles (10% weight/volume, 0.7-0.9 micron (available fromSeradyn, Indianapolis, Ind.)) was mixed with 788 μl of coating buffer(MES buffer, pH 6.5 with SDS) for approximately 10 minutes at roomtemperature. Then, 112 μl of an HCV p9MB-18 antigen solution (0.89mg/ml) was added to the microparticle solution, mixed and tumbled for 16hours at room temperature. The microparticles were pelleted at 12,000×gfor 10 minutes in an Eppendorf microfuge. The suspension was removed,and the microparticles were washed with wash buffer (phosphate, NaCl,EDTA, SDS, pH 6.5) and heat stressed at 56° C. for 16-20 hours. Themicroparticles were then resuspended in 500 ul of microparticle diluent(Phosphate Buffer, pH 6.5, EDTA, NaCl, Sucrose, SDS, azide) at a finalconcentration of 2.0%.

[0123] iv. Blending of C11-14 mAb Coated Microparticles with HCVp9MB-18-Coated Microparticles and HCV c200 Antigen-CoatedMicroparticles. Fifty microliters of p9MB-18 coated microparticles (2%solids stock) prepared as in Example (V) (D) (iii), 62.5 μl of HCV c200Ag coated microparticles (2% solids stock) prepared as described inExample (V) (D) (ii) and 125 μl HCV C11-14 microparticles (2% solidsstock) prepared as described in Example (V) (B1) (i) were mixed with4762.5 μl microparticle diluent (EDTA, SDS, Sucrose and Proclin, PBS pH6.5).

[0124] Sensitivity: The seroconversion sensitivity was 95.8% as comparedto nucleic acid testing data. The PRISM® HCV Ag/Ab Real Combo assaydetected 23/24 positive bleeds as reactive. Data is summarized in TableV, below. Overall sensitivity for the seroconversion panels shown inTable IV are comparable between the blended microparticles prepared asdescribed in Example (V) (C) (i) and microparticles prepared asdescribed in Example (V) (C) (iv) using assay format provided in FIG. 3.However, for anti-HCV Core, specific sample P9MB18-coated microparticlesshowed significant improvement over core specific peptide (11-28 aa,refer to Table VI), when used in combination in combo assay format(Table VI).

[0125] D. Preparation of p9MB31 Coated Microparticles:

[0126] i. A 100 μl aliquot of microparticles (10% weight/volume, 0.7-0.9micron, available from Seradyn, Indianapolis, Ind.) was mixed with 788μl of coating buffer (MES buffer, pH 6.5 with SDS) for approximately 10minutes at room temperature. Then, 112 μl of an HCV p9MB-31 antigensolution (0.89 mg/ml) was added to the microparticle solution, mixed,and tumbled for 16 hours at room temperature. The microparticles werepelleted at 12,000×g for 10 minutes in an Eppendorf microfuge. Thesuspension was removed, and the microparticles were washed with washbuffer (Phosphate, NaCl, EDTA, SDS, pH 6.5) and heat stressed at 56° C.for 16-20 hours. The microparticles were then resuspended in 500 μl ofmicroparticle diluent (Phosphate Buffer, pH 6.5, EDTA, NaCl, Sucrose,SDS, azide) at a final concentration of 2.0%.

[0127] Sensitivity: The sensitivity of the HCV panels was comparedbetween microparticles coated with p9MB-18 and p9MB-31 r-antigen usingthe assay format provided in FIG. 1. Overall, sensitivity for both ofthese r-antigens is comparable as shown in Table IV. TABLE IV p9MB18coated p9MB31 coated uPS (V) (D) (iv) uP (V) (E) (i) Panel or SampleS/CO S/CO Panel A 13.03 9.43 NABI #2 21.9 27.76 NABI #15 28.21 33.04

[0128] TABLE V HCV RNA HCV Ab/Ag Ag earlier Day Test HCV Ab HCV Ag RealCombo Geno-type than Ab by Number Results S/CO S/CO S/CO 1a 38 Days 0 −0.09 0.49 0.39 24 + 0.11 10.63 2.47 27 + 0.10 43.77 4.66 31 + 0.11 72.928.09 62 + 5.19 44.41 5.61 64 + 5.22 69.55 5.86 69 + 5.91 12.92 4.22 71 +6.29 7.09 3.77 1b 18 Days 0 + 0.09 76.61 6.21 4 + 0.09 56.09 4.29 7 +0.08 39.63 2.97 13 + 0.34 32.14 2.44 18 + 1.53 14.93 1.68 21 + 3.2019.97 2.64 164 Not Tested 5.86 0.61 3.61 1a 23 Days 0 − 0.09 0.45 0.39 2− 0.08 0.45 0.42 17 + 0.07 20.06 1.58 19 + 0.09 45.84 4.11 24 + 0.0981.03 6.26 26 + 0.07 63.30 5.95 36 + 0.31 74.78 8.44 40 + 4.03 49.535.74 1a 32 Days 0 − 0.46 0.54 0.29 22 − 0.42 0.53 0.43 24 − 0.43 0.470.40 42 + 0.46 8.79 0.99 46 + 0.44 22.26 2.02 74 + 4.22 19.82 2.40 76 +4.50 23.78 2.99

[0129] TABLE VI Peptide blended Combo ups P9MB-18 blended Combo upsPanel or (V) (C) (i) (V) (D) (iv) sample S/CO S/CO Panel A 0.95 1.25NABI #7 0.44 2.27 NABI 1.15 2.04 0141044662

EXAMPLE VI Preparation of Acridinium-Labeled Conjugates

[0130] A. Conjugate for Antibody Assay:

[0131] For the antibody assay, either mouse anti-human IgG directlylabeled with acridinium or a pre-complex of biotinylated anti-human F(ab′)₂ and acridinium anti biotin conjugate was used.

[0132] i. Pre-complex of biotinylated anti-human F(ab′)₂ and acridiniumanti-biotin conjugate. The labeled anti-biotin antibody was prepared asdisclosed in U.S. Pat. No. 5,705,330. The pre-complex of biotinylatedAnti-human F(ab′)₂ and acridinium anti-biotin conjugate were alsoprepared as disclosed in U.S. Pat. No. 5,705,330.

[0133] ii. Acridinium labeled Mouse anti-Human IgG. Fifty-three pointsix microliters of conjugation buffer (CB) containing sodium phosphate,NaCl, 3-(3-chlolamidopropyl)-dimethylammonio-1-propane-sulfonate (CHAPS,Sigma Chemical Company, Saint Louis, Mo), pH 8.0 and 7.2 μl ofN-hydroxysuccinimide ester of10-(3-sulfopropyl)-N-tosyl-N-(2-carboxyethyl)-9-acridinium carboxamide(4 mg/ml in dimethyl formamide) was added to 131 μl of Mouse anti-HumanIgG (4.59 mg/ml) and 601 μl of PBS at room temperature. The reactionmixture was mixed with a rotator for 20 minutes at room temperature. Thereaction was quenched by loading the reaction mixture onto the HPLC.This was applied to a 300×7.8 mm Bio-Sil SEC-250 gel filtration column(Bio-Rad, Richmond, California) which had been equilibrated with buffercontaining CHAPS, NaCl and sodium phosphate, pH 6.3. The column waseluted at 1.0 ml/min with the same buffer using a Beckman 421Acontroller equipped with a model 114M pump. Fractions of 1 ml werecollected, and the absorbance determined at 280 nm and 370 nm with aBeckman DU-7 spectrophotometer. The extent of acridinium incorporationwas calculated as described in U.S. Pat. No. 5,705,330. The acridiniumto IgG ratio (mole/mole) obtained was approximately 2.5. The conjugatewas stored at 4° C.

[0134] B. Conjugate for Antigen Assay:

[0135] i. Acridinylation of c11-10 conjugate. A similar procedure asmentioned in Example (V) (A) (ii) was used except for the followingchanges. Seven hundred microliters of conjugate buffer, 300 ul (1 mg/ml)of C11-10 Mab and 2.9 ul (4 mg/ml) of acridinium derivative were mixedfor 10 minutes at room temperature. The acridinium to IgG ratio(mole/mole) obtained was approximately 2.0. The conjugate was stored at4° C.

[0136] C. Conjugate for Combo Assay:

[0137] Blending of Acridinylated Mouse anti-Human IgG and AcridinylatedC11-10 Mab Conjugate:

[0138] Fourteen microliters of Acridinylated Mouse Anti-Human IgG (1ug/ml) was mixed with 390 μl of Acridinylated C11-10 Mab (1.79 μg/ml)conjugate yielding 2 ng/ml Mouse anti-Human IgG with 100 ng/ml C11-10,incubated for 2 hours and filtered before use. Preparation of Mouseanti-Human IgG and Acridinylated C11-10 Mab conjugate are described inExample (V) (A) (ii) and (VI) (B), respectively.

EXAMPLE VII Detection of HCV Core Protein by Monoclonal Antibodies

[0139] Since a large number of anti-HCV core monoclonal antibodies wereavailable for use in developing an antigen detection assay, it wasnecessary to determine which combination of monoclonal antibodies wouldprovide the greatest sensitivity. Because the number of combinationspossible when using more than one monoclonal on the solid phase (i.e.for capture) and in the liquid phase (i.e. detection) is extremelylarge, a simplified “screening” method was used to identify the bestperforming pair of monoclonals. It was assumed that once the mostsensitive pair was identified, other monoclonals could be added toimprove assay sensitivity, if necessary.

[0140] In order to identify the best pairs, therefore, monoclonalantibodies were coated onto microparticles or conjugated with acridiniumas described in Example IV and V. Screening assays used monoclonalantibody-coated microparticles (0.40 μm diameter) at a workingconcentration of 0.09-0.15% solids and conjugated monoclonals at aworking concentration of 100-125 ng/ml. For all experiments, the samepositive and negative control plasma were used (0.1 ml for each assay).The positive control serum was from an HCV-infected individual whotested negative for HCV antibodies but whose plasma had an HCV RNA titerof 19,000,000 copies per ml. The negative control plasma was from anormal blood donor who was negative for HCV antibodies and RNA. Assayswere performed using the instrumentation and operation methods asdescribed in Example IX.

[0141] Table VII shows the mean signal-to-negative (S/N) values obtainedupon testing the various pairs of monoclonal antibodies for theirability to detect HCV core antigen in the positive control human plasma(nd: not determined). From this data, it is apparent that some pairs ofmonoclonal antibodies exhibit greater sensitivity than others and thatthe sensitivity was dependent upon the proper configuration of theassay. For example, when monoclonal antibody A05 was used as the capturereagent and C11-10 was used as the detection reagent, the resulting S/Nvalue was 150.0; however, when the opposite configuration was used, theresulting S/N value was only 6.8 TABLE VII Detection of HCV Core Antigenin Human Plasma by Various Pairs of Anti-core Monoclonal AntibodiesCONJUGATE 107-35- 14-635- 14-726- 13-975- 13-959- 14-178- 14-886-110-81- 14-945- C11- C11- C11- Mab 54 225 217 157 270 125 216 17 218C11-3 C11-7 10 14 15 Mab MICRO- C11- C11- C11- PARTICLE A04 A05 A06 A08A09 A13 A14 A15 A218 C11-3 C11-7 10 14 15 14-1708-403 A01 0.6 nd 1.3 2.51.0 1.3 5.2 1.0 1.4 2.4 0.9 53.0 1.9 3.5 14-153-462 A02 nd nd 1.4 nd 0.91.5 1.5 nd 2.4 0.6 1.0 22.0 1.8 0.2 14-1269-281 A03 0.8 nd 1.3 2.4 1.11.3 7.4 0.9 1.4 2.2 2.9 101.6 3.0 6.0 107-35-54 A04 nd nd 0.9 nd nd ndnd nd nd 1.7 1.2 7.6 12.9 1.3 14-635-225 A05 1.2 nd 1.5 2.9 0.9 1.0 8.41.0 1.5 4.2 2.1 150.0 1.9 4.0 14-726-217 A06 nd nd 0.6 nd 1.6 1.8 2.5 nd2.7 1.7 2.4 9.5 3.8 0.7 13-975-157 A08 nd nd 0.7 nd nd nd nd nd nd 1.40.8 4.6 1.6 3.2 13-959-270 A09 nd nd 1.6 nd nd nd nd nd nd 1.5 1.2 2.41.5 1.0 14-1705-255 A10 nd nd 0.7 nd 1.3 1.7 1.2 nd 2.5 1.6 1.3 13.6 4.51.2 14-188-104 A11 nd nd 0.8 nd 1.0 1.2 2.8 nd 1.2 2.8 0.7 16.5 1.8 2.414-1287-252 A12 nd nd 0.9 nd nd nd nd nd nd 2.8 1.9 6.3 2.4 6.114-886-216 A14 nd nd 0.9 nd nd nd nd nd nd 3.2 3.1 3.0 4.2 2.1 110-81-17A15 nd nd 1.5 nd nd nd nd nd nd 1.0 1.9 6.3 2.4 2.3 14-947-104 A16 0.5nd 1.1 6.1 1.5 1.3 6.1 1.1 1.1 1.6 1.4 69.0 4.7 1.2 C11-3 C11-3 0.6 4.7nd nd 1.1 2.1 1.0 nd 0.9 2.0 1.1 11.1 4.6 3.6 C11-7 C11-7 1.2 3.9 nd nd1.1 0.8 3.6 nd 1.3 2.5 1.8 6.2 3.4 2.0 C11-10 C11-10 0.6 6.8 nd nd 0.91.4 1.1 nd 1.0 8.2 1.9 4.3 4.6 2.8 C11-14 C11-14 0.8 2.0 1.5 nd 0.8 1.113.4 nd 1.7 7.9 0.9 208.0 2.2 14.4 C11-15 C11-15 1.6 4.6 nd nd 1.2 1.41.3 nd 1.5 5.1 1.5 4.9 3.8 5.7

EXAMPLE VIII HCV Core Antigen Assay Sample Diluent Buffer

[0142] The HCV core antigen assay for PRISM®, as described in ExampleXI, utilizes a sample diluent buffer (SDB) for dilution of the humanserum or plasma sample to be tested. The monoclonal antibody-coatedmicroparticles are then added to form a reaction mixture. It is possiblethat the sensitivity and specificity of the antigen detection assay isaffected by the composition of the SDB, in terms of the ingredients andtheir concentration.

[0143] It was hypothesized that, since HCV is believed to be anenveloped virus, it would be necessary to include detergent (surfactant)in the SDB to remove the lipid envelope, thereby exposing the coreprotein to solution. In addition, it was surmised that addition ofchaotropic salts to the SDB might aid in dissolution of the nucleocapsidcomplex which could enhance detectability of core antigen.

[0144] To investigate the possible effects of SDB composition on the HCVcore antigen assay sensitivity, a series of buffers was prepared andtested in an HCV core antigen assay comprised of monoclonal antibodyC11-7 or C11-14 coated microparticles (as stated in the table legends)and acridinium labeled monoclonal antibody C11-10 conjugate. Thesimplest SDB used (also referred to as basal buffer), in terms of numberof components, consisted of 0.1 M potassium phosphate, pH 7.2, 10 mMEDTA. This is the buffer to which detergents and salts were added. Theperformance of the SDBs was determined by examining their effect on thesignal-to-negative (S/N) ratio obtained upon testing of a positivecontrol human plasma from an individual who tested negative for HCVantibodies but whose plasma had an HCV RNA titer of 19,000,000 copiesper ml. The negative control plasma was from a normal blood donor whowas negative for HCV antibodies and RNA. Screening assays used coatedmicroparticles at a working concentration of 0.09-0.15% solids andC11-10 conjugate at a working concentration of 100-125 ng/ml. For allexperiments, the same positive and negative control plasma was used (0.1ml for each assay). Assays were performed using the instrumentation andoperation methods as described in Example VIII.

[0145] As shown in Table VIII, the S/N value obtained varies greatlydepending upon the detergent added to the sample diluent buffer and itsconcentration. Addition of the zwitterionic surfactant SB-12 (laurylsulfobetaine) gave the highest S/N values. In addition, as shown inTable IX, the highest S/N values were again seen with SB-12 whencompared to other zwitterionic detergents of the same class but withdifferent alkyl chain lengths. Titration of the amount of SB-12 added tothe basal buffer in the presence of 0.5% or 2% Triton X-100 is shown inTable X. Increasing the SB-12 concentration over 6% diminished S/Nvalues obtained in the core antigen assay significantly.

[0146] Further experiments examined the effect of the addition of saltsor different combinations of zwitterionic or nonionic detergents to thesensitivity of the core antigen assay. Results presented in Tables XIand XII suggest a marginal effect on S/N is observed when KCl issubstituted for NaCl, the same is true for the addition of urea. Thesample diluent buffers containing SB-16 (palmityl sulfobetaine) appearto exhibit enhanced S/N values. The effect of urea was examined byincluding increasing concentrations in one of the SDBs that gavereasonably high S/N values compared to the others in a previousexperiment (Table XIII). In this particular buffer, addition of urea toa final concentration of 2.0-2.5 M appears to have increased S/N valuesmost significantly.

[0147] The effect on S/N values by the addition of various proteins orserum from nonhuman sources to a sample diluent buffer was also examined(Table XIV). The inclusion of bovine serum albumin, with or withoutother proteinacious components, only marginally increased the S/N valuesobtained upon testing of the HCV positive control serum. In contrast,some combinations of protein or sera actually decreased the S/N valuerelative to that observed for the protein-free buffer. TABLE VIII Effectof Detergent on HCV Core Antigen Detection Detergent/Additive (in basalbuffer) Acronym S/N @ 0.5% S/N @ 2% Dodecyldimethyl-3-amonio-propaneSB-12 2.4 7.9 sulfonate 1-dodecylpyridinium chloride DPC 0.1 6.9 Sodiumdodecylsulfate SDS 2.7 5.0 Cholamidopropyldimethylamonio CHAPS 1.0 4.7propanesulfonate 3a, 7a, 12a-Trihydroxy-5b-cholanic acid Cholate 1.2 2.2t-Octylphenoxypolyethoxyethanol Triton X-100 2.0 1.8Carboxymethyltrimethylammonium Betaine 1.9 1.5 Taurocholic acid TCA 1.11.3 Dodecyltrimethylammonium bromide DTAB 0.2 1.1 Mixture of steroids,polysacc., Saponin 3.1 0.9 detergents N-Dodecanoyl-N-methylglycine(N-lauroyl NLS 1.6 0.2 sarcosine) Cetyltrimethylammonium bromide CTAB1.6 nd Tetradecyltrimethylammonium bromide TDTAB 1.6 nd

[0148] TABLE IX Detergent/Additive (in basal buffer) Acronym S/N @ 0.5%S/N @ 2% (3-[(3-cholamidopropyl)dimethylammonio]- CHAPSO 2.9 11.52-hydroxy-1-propanesulfonate N-dodecyl-N,N-(dimethylammonio)butyrateDDMAB 1.9 nd N-dodecyl-N,N- DDMAU 4.1 9.4 (dimethylammonio)undecanoateN,N-dimethyldodecylamine-N-oxide LDAO 3.8 1.8N-octyl-N,N-dimethyl-3-ammonio-1- SB-8 2.5 3.8 propanesulfonateN-decyl-N,N-dimethyl-3-ammonio-1- SB-10 3.0 5.2 propanesulfonateN-dodecyl-N,N-dimethyl-3-ammonio-1- SB-12 16.0 38.3 propanesulfonateN-tetradecyl-N,N-dimethyl-3-ammonio-1- SB-14 4.5 0.8 propanesulfonateN-hexadecyl-N,N-dimethyl-3-ammonio-1- SB-16, 0.125% 5.0 ndpropanesulfonate

[0149] TABLE X Detergent/Additive S/N with S/N with (in basal buffer)0.5% Triton X100 2% Triton X100 2% SB-12 13.3 14.8 4% SB-12 14.3 16.6 6%SB-12 10.7 15.3 8% SB-12 0.8 0.91

[0150] TABLE XI SDB Components and Final Concentration Triton Exp 1 Exp2 NaCl SB-12 SB-16 CTAB X-100 Urea S/N S/N (no 3.85 nd buffer added)Water 3.41 4.7 0.5 M 2.84 6.7 0.5 M 1.80% 32.17 34.2 0.5 M 2% 93.4 78.20.5 M 2% 2.5 M 106.3 82.9 0.5 M 2% 0.10% 97.1 67.4 0.5 M 2% 0.10% 2.5 M93.9 86.3 0.5 M 2% 0.10% 1.80% 98.8 79.1 0.5 M 2% 0.10% 1.80% 2.5 M 84.890.5 0.5 M 2% 2.5 M 105.2 92.9 0.5 M 2% 0.10% 106.8 108.1 0.5 M 2% 0.10%2.5 M 142.2 102.3 0.5 M 2% 0.10% 1.80% 115.1 101.6 0.5 M 2% 0.10% 1.80%2.5 M nd 103.0

[0151] TABLE XII SDB Components and Final Concentration KCl SB-12 SB-16CTAB Triton X-100 Urea S/N 0.5 M 2% 48.6 0.5 M 2% 2.5 M 86.8 0.5 M 2%0.10% 52.8 0.5 M 2% 0.10% 2.5 M 77.6 0.5 M 2% 0.10% 1.80% 83.6 0.5 M 2%0.10% 1.80% 2.5 M 106.8 0.5 M 2% 2.5 M 122.6 0.5 M 2% 0.10% 2.5 M 1360.5 M 2% 0.10% 1.80% 127.9 0.5 M 2% 0.10% 1.80% 2.5 M 113.7

[0152] TABLE XIII Effect of Urea on Antigen Assay Sensitivity HCVpositive S/N values at various urea concentrations control plasma 0.00.5 1.0 1.5 2.0 2.5 3.0 5.0 dilution factor M M M M M M M M 1:2 35.231.3 32.9 33.4 43.0 34.6 33.2 1.1 1:4 19.9 15.7 17.7 13.6 21.5 18.9 15.61.1 1:8 9.1 8.2 9.3 6.1 10.5 8.1 8.0 0.9 1:16 5.9 4.3 5.7 5.3 6.6 5.65.5 1.0 1:32 3.8 3.2 3.6 3.3 3.6 3.5 3.0 1.0

[0153] TABLE XIV Effect of Protein or Serum on Antigen Assay SensitivityS/N S/N Component(s) Added (final conc., w/v)y PC 1:2 PC 1:16 Noadditions 69.3 9.6   1% BSA, 2% mouse serum 73.7 10.3   1% BSA, 0.1%casein 70.5 10.7   1% BSA 70.2 10.3   3% horse serum 68.2 9.6   2% mouseserum 65.6 10.9 0.1% casein 51.7 8.1   2% mouse serum, 0.1% casein 50.97.9   1% BSA, 3% horse serum 50.6 8.8 0.1% casein, 3% horse serum 40.76.9   2% mouse serum, 3% horse serum 34.3 4.9

EXAMPLE IX PRISM® HCV Ab, PRISM® HCV Ag, and PRISM® HCV Ab/Ag ComboAssays

[0154] The PRISM® antibody assay is described in U.S. Pat. No.5,705,330, incorporated herein by reference and the PRISM® antigen andantibody assays are described in Shah and Stewart, The ImmunoassayHandbook, second edition, edited by David Wild, p 297-303 (2001), alsoincorporated herein by reference.

[0155] With respect to the present invention, the following procedureswere utilized:

[0156] HCV Ab Assay:

[0157] Assay format is provided in FIG. 1.

[0158] Generally, at station 1, 50 μl of control or sample, 50 μl ofspecimen diluent buffer (SDB, Phosphate buffer, pH 7.0 containing Tween20, newborn calf serum, NaCl, superoxide dismutase (SOD), E. coli lysateand azide), and 50 μl of HCV antigen coated microparticles (prepared asdescribed in Example (V) (A1) (iv) above) were dispensed into eachincubation well and assay timing was started. These were mixed by mutualdiffusion of each into the other without external agitation or shakingto form a reaction mixture. At station 4, the reaction mixture wastransferred to a detection well which contained a fibrous matrix andwashed twice with 300 μl of transfer wash (TW, containing borate buffer,pH 7.0, with NaCl, Tween −20, Glycerol and Proclin 300). After 18minutes of incubation at room temperature, 50 μl of a pre-complexedbiotinylated F(ab′)₂/acridinium labeled anti-biotin, (biotinylatedF(ab′)₂ fragment of goat anti-human IgG and acridinium labeledanti-biotin antibody), was dispensed into the matrix of the detectionwell at station 5. The well was incubated for 23 minutes at 37° C., andthe fibrous matrix containing the reaction mixture was washed threetimes with 100 μl of FW, containing MES (2-[N-morpholino] ethanesulfonicacid), pH 5.7, with NaCl and Proclin 300 at station 8. At station 9, asin all of the assays described below, a chemiluminescence (CL) signalwas generated by addition of an alkaline hydrogen peroxide solution, andthe photons were measured by a photo multiplier tube. The amount oflight emitted is proportional to the amount of antibody in the sample.The presence or absence of antibody in the sample is determined bycomparing the number of photons collected from the sample to a cutoff(S/CO) value determined from a calibration performed in the batch. Theresults are expressed as S/CO (signal to cutoff) in Table XV below. Thecutoff value is calculated by the sum of product of the averagechemiluminescence counts of the positive control (n=4) times 0.55 plusthe average chemiluminescence counts of the negative control (n=4).

[0159] Sensitivity: The seroconversion sensitivity was 100% as comparedto the HCV RNA data provided in vendor certificate of analysis data forthe selected seroconversion panels tested. Data is summarized in TableXV.

[0160] Specificity: Based on repeat reactive rates, the specificity ofthe HCV Ab assay was >99% with the population tested (Table XVII).

[0161] HCV Ag Assay:

[0162] Assay format is provided in FIG. 2.

[0163] Mab C11-14/Mab C11-10 Pair:

[0164] Generally, at station 1, 100 μl of control or sample, 50 μl ofspecimen diluent buffer (SDB, Sodium phosphate, EDTA, Triton X-100, Ureaand sodium azide), and 50 μl of HCV Mab coated microparticles (preparedas described in Example (V) (B1) (i)) were dispensed into eachincubation well and the assay timing was started. These were mixed bymutual diffusion of each into the other without external agitation orshaking to form a reaction mixture. At station 4, the reaction mixturewas transferred to a detection well which contained a fibrous matrix andwashed twice with 300 μl of transfer wash (TW) (MES, NaCl, Triton X-100,PEG, Antifoam, Proclin 300, pH 5.6) after 18 minutes of incubation atroom temperature. At station 5, 50 μl of acridinylated C11-10 Mabconjugate (as mentioned in Example (VI) (B)) was dispensed into thematrix of the detection well. The contents of the well were incubatedfor 23 minutes, and the fibrous matrix containing the reaction mixturewas washed one time with 200 μl of final wash (FW) (Tris buffer withLiCl and LDS) followed by three times with 100 ul of FW. The CL signalwas triggered and measured at station 9. The results are expressed asS/CO (signal to cutoff) in Table XV. The cutoff value is 2.2 times theaverage chemiluminescence count of the negative control (n=5).

[0165] Sensitivity: Two groups of commercially available seroconversionpanels containing serially collected samples from individuals whodeveloped antibodies to HCV were tested in the prototype PRISM® HCVantigen test and in the PRISM® HCV antibody test. For the first group ofseroconversion samples, the first available bleed date was negative forHCV RNA. In subsequent bleed dates, HCV RNA was detected for one or morebleed dates, followed in all cases by detection of antibodies to HCV.For the second group of seroconversion panels, the first bleed date wasalready positive for HCV RNA; antibodies to HCV were detected insubsequent bleed dates. For the two groups seroconversion sensitivitywas 98.5% as compared to data obtained by HCV RNA testing. The PRISM®HCV Ag detected 67/68 HCV RNA positive bleeds as reactive. Data issummarized in Table XVI. These data indicate that HCV Ag testing detectsHCV infection in individuals who have not yet mounted an antibodyresponse.

[0166] Specificity: Based on repeat reactive rates, the specificity ofthe HCV Ag assay was >99% with the population tested (Table XVIII).

[0167] HCV Ag Assay: Mab A5 (14-635-225)/Mab C11-10 Pair:

[0168] An assay procedure, similar to that mentioned for C11-14/C11-10,was used. The only difference was that the test used Abbott A5(14-635-225) Mab coated microparticles instead of Mab C11-14 coatedmicroparticles.

[0169] Sensitivity: A total of 4 seroconversion panels were evaluatedand sensitivity was compared with the data generated using C11-14/C11-10pair. Both these pairs detected the same number of positive bleeds.Sensitivity data for A5 (14-635-225)/C11-10 pair is summarized in TableXVI.

[0170] Specificity: Based on repeat reactive rates, the specificity ofthe HCV Ag assay was 100% with the mini population (n=100) tested (TableXIX).

[0171] HCV Ag Assay: Mab C11-14 and C11-3/Mab C11-10 Pair:

[0172] An assay procedure, similar to that mentioned for C11-14/C11-10,was used. The only difference was the use of C11-14 and C11-3 blendedmicroparticles (Example (IV) (B1) (iv)) instead of Mab C11-14 coatedmicroparticles.

[0173] Sensitivity: The performance of this pair was assessed bycomparing the S/N ratio against panels consisting of recalcified humanplasma positive for HCV Core antigen (termed “PC”) and a human plasmanegative for HCV antigens and antibodies (termed “NC”) (Table XX). TheS/N was determined by the formula:

S/N=Average of PC/Average of NC

[0174] The average chemiluminescence counts of four specimens were usedto determine each average.

[0175] PRISM® HCV Ag/Ab Combo Assay:

[0176] Two different formats (i.e., Dual Combo assay and Real comboassay) were evaluated on the PRISM® HCV Ag/Ab Assay as follows:

[0177] Dual Combo Assay: The HCV Ag/Ab dual combo assay is runsimultaneously on PRISM® using two different channels. A total of sixchannels in PRISM® are used simultaneously to run several assays (HIV,HBcore, HBsAg, HTLV, and HCVAb) five of which are currently in use,while one channel remains open for new markers (e.g. HCV Ag assay) orcan be reserved in case one of the channels become problematic. Thus, byusing one channel for an HCV Ag assay and five other channels for fiveother assays, a reserve channel is not available for use.

[0178] The PRISM® HCV Ab and PRISM® HCV Ag assays were performedindividually. The results from both assays were combined to produce asingle, final report.

[0179] Real Combo Assay: The PRISM® HCV Ag and HCV Ab assays werecombined and performed as a single assay in one of the PRISM® channels.

[0180] PRISM® HCV Ag/Ab Dual Combo Assay:

[0181] Sensitivity: The seroconversion sensitivity of the HCV Ab/Ag DualCombo assay was 98.5%. Data is summarized in Table XV.

[0182] Specificity: Based on repeat reactive rates, the specificity ofthe HCV Ab/Ag Dual Combo assay was >99% with the population tested(Table XXI).

[0183] PRISM® HCV Ag/Ab Real Combo Assay:

[0184] Assay format is provided in FIG. 3. The 2 step PRISM® HCV Comboassay was performed as mentioned above for the HCV Ab or Ag assay withthe following changes: At station 1, 100 μl of control or sample, 50 μlof specimen diluent buffer (Phosphate buffer, pH 7.0 containing Tween20, newborn calf serum, NaCl, Tween-20, superoxide dismutase (SOD), E.coli lysate and azide), and 50 μl of HCV antigen and Mab blendedmicroparticles (prepared as described in Example (V) (C) (i) above) weredispensed into each incubation well and the assay timing was started. Atstation 4, the reaction mixture was transferred to a detection wellwhich contained a fibrous matrix and washed twice with 300 ul oftransfer wash (MES, NaCl, Triton X-100, PEG, Antifoam, Proclin 300, pH5.6). After 18 minutes of incubation at 37 degree C., 50 ul of blendedconjugate acridinylated C11-10 and Acridinylated Mouse anti-Human IgG(prepared as described in Example (VI) (C)) was dispensed into thematrix of the detection well at station # 5. The well was incubated for23 minutes, and the fibrous matrix containing the reaction mixture waswashed three times with 100 μl of final wash (Tris buffer with LiCl andLDS). The CL signal was triggered and measured at station 9. The resultsare expressed as S/CO (signal to cutoff) in Table XV below. The cutoffvalue is 2.2 times the average chemiluminescence count of the negativecontrol (n=3).

[0185] Sensitivity: The seroconversion sensitivity was 95.8% as comparedto PCR data. The PRISM® HCV Ag/Ab Real Combo assay detected 23/24positive bleeds as reactive. Data is summarized in Table XVI.

[0186] Specificity: Based on repeat reactive rates the specificity ofHCV Ag/Ab Real Combo assay was 100% with the population tested (TableXX): TABLE XV HCV Ab/Ag HCV RNA Dual Ag earlier Day Test HCV Ab HCV AgCombo Genotype than Ab by Number Results S/CO S/CO S/CO 1a 38 Days 0 −0.09 0.49 NEG 24 + 0.11 10.63 POS 27 + 0.10 43.77 POS 31 + 0.11 72.92POS 62 + 5.19 44.41 POS 64 + 5.22 69.55 POS 69 + 5.91 12.92 POS 71 +6.29 7.09 POS 1a 20 Days 0 + 0.10 107.78 POS 3 + 0.11 97.81 POS 10 +0.15 63.52 POS 20 + 1.57 Not POS Tested 1b 18 Days 0 + 0.09 76.61 POS4 + 0.09 56.09 POS 7 + 0.08 39.63 POS 13 + 0.34 32.14 POS 18 + 1.5314.93 POS 21 + 3.20 19.97 POS 164 Not Tested 5.86 0.61 POS 2b 14 Days0 + 0.10 8.41 POS 2 + 0.40 23.62 POS 7 + 2.62 14.09 POS 9 + 3.09 16.67POS 14 + 3.99 4.74 POS 1a 42 Days 0 − 0.09 0.76 NEG 2 − 0.08 0.47 NEG 7− 0.09 0.41 NEG 9 − 0.09 0.40 NEG 15 − 0.08 0.42 NEG 17 − 0.08 0.49 NEG22 − 0.07 0.49 NEG 24 − 0.08 0.45 NEG 29 − 0.09 0.52 NEG 31 − 0.08 0.51NEG 36 − 0.08 0.57 NEG 38 − 0.09 0.51 NEG 43 − 0.08 0.52 NEG 45 − 0.090.43 NEG 50 − 0.09 0.69 NEG 52 − 0.08 0.52 NEG 57 − 0.09 0.49 NEG 64 −0.09 0.90 NEG 67 − 0.08 0.52 NEG 74 − 0.09 0.51 NEG 79 − 0.08 0.52 NEG84 − 0.09 0.40 NEG 105 − 0.09 0.45 NEG 108 − 0.08 0.66 NEG 112 − 0.090.57 NEG 119 − 0.09 0.45 NEG 121 − 0.09 0.42 NEG 140 + 0.12 10.65 POS143 + 0.09 3.81 POS 147 + 0.10 9.30 POS 150 + 0.09 34.08 POS 154 + 0.0958.01 POS 157 + 0.09 80.90 POS 161 + 0.08 107.11 POS 164 + 0.09 114.41POS 168 + 0.09 93.29 POS 171 + 0.10 89.06 POS 182 + 1.83 63.62 POS 186 +4.39 68.72 POS 189 + 5.20 119.62 POS 1a 21 Days 0 + 0.08 63.86 POS 4 +0.07 50.76 POS 17 + 0.09 73.66 POS 21 + 1.02 45.58 POS 25 + 4.03 60.94POS 29 + 5.08 47.38 POS 1a 37 Days 0 + 0.08 33.66 POS 2 + 0.06 30.83 POS7 + 0.08 30.10 POS 9 + 0.07 39.66 POS 26 + 0.07 25.51 POS 32 + 0.1215.29 POS 37 + 2.43 15.51 POS 41 + 3.36 3.10 POS 1a 28 Days 0 + 0.0967.75 POS 2 + 0.09 87.93 POS 10 + 0.10 36.53 POS 12 + 0.10 60.67 POS19 + 0.10 39.62 POS 21 + 0.11 26.25 POS 28 + 2.78 9.94 POS 30 + 4.0017.02 POS 35 + 4.71 15.26 POS 37 Not tested 4.84 13.02 POS 1a 25 Days0 + 0.15 4.73 POS 2 + 0.40 6.63 POS 8 + 0.16 7.48 POS 10 + 0.11 5.20 POS16 + 0.17 7.60 POS 18 + 0.11 7.58 POS 23 + 0.64 8.66 POS 25 + 2.11 9.58POS 30 + 2.76 6.21 POS 32 + 3.39 7.84 POS 49 + 6.12 1.83 POS 53 + 6.131.93 POS 56 + 6.34 1.63 POS 1a 28 Days 0 − 0.13 1.27 POS 2 − 0.23 0.52NEG 8 − 0.09 0.50 NEG 11 + 0.10 0.54 NEG 15 + 0.10 1.92 POS 18 + 0.111.90 POS 28 + 0.12 2.42 POS 30 + 0.10 7.04 POS 35 + 0.14 6.01 POS 37 +0.98 13.68 POS 43 + 4.74 10.07 POS 46 + 5.27 4.91 POS 1a 13 Days 0 +0.10 1.82 POS 3 + 0.10 1.72 POS 5 + 0.15 1.35 POS 11 + 0.97 1.70 POS13 + 1.26 3.63 POS 19 − 3.70 2.94 POS 25 − 4.89 2.43 POS 27 − 5.20 1.61POS 32 − 5.61 1.35 POS 35 − 5.86 1.30 POS 41 − 6.11 0.88 POS 45 − 5.690.67 POS 48 − 5.95 1.94 POS 1a 23 Days 0 − 0.09 0.45 NEG 2 − 0.08 0.45NEG 17 + 0.07 20.06 POS 19 + 0.09 45.84 POS 24 + 0.09 81.03 POS 26 +0.07 63.30 POS 36 + 0.31 74.78 POS 40 + 4.03 49.53 POS 1a 33 Days 0 −0.09 0.54 NEG 3 − 0.08 0.50 NEG 7 − 0.08 0.58 NEG 12 − 0.09 0.54 NEG 14− 0.09 0.56 NEG 19 − 0.10 0.53 NEG 25 − 0.10 0.51 NEG 28 − 0.09 0.50 NEG32 − 0.09 0.50 NEG 35 − 0.10 0.35 NEG 39 − 0.09 0.53 NEG 45 + 0.10 9.43POS 47 + 0.11 42.00 POS 52 + 0.11 28.05 POS 56 + 0.11 25.63 POS 60 +0.09 78.15 POS 73 + 0.18 9.54 POS 78 + 1.83 5.34 POS 80 + 2.13 3.40 POS1a 32 Days 0 − 0.46 0.54 NEG 22 − 0.42 0.53 NEG 24 − 0.43 0.47 NEG 42 +0.46 8.79 POS 46 + 0.44 22.26 POS 74 + 4.22 19.82 POS 76 + 4.50 23.78POS 3a 141 Days 0 + 0.12 2.81 POS 4 + 0.49 1.95 POS 11 + 2.48 1.41 POS13 + 2.54 1.41 POS 44 + 4.29 0.43 POS 46 + 4.68 0.43 POS 1a 38 Days 0 −0.09 0.49 0.38 24 + 0.11 10.63 1.60 27 + 0.10 43.77 5.32 31 + 0.11 72.9211.04 62 + 5.19 44.41 10.95 64 + 5.22 69.55 10.16 69 + 5.91 12.92 4.5171 + 6.29 7.09 3.68 1b 18 Days 0 + 0.09 76.61 14.09 4 + 0.09 56.09 5.597 + 0.08 39.63 4.75 13 + 0.34 32.14 3.81 18 + 1.53 14.93 4.16 21 + 3.2019.97 3.81 164 Not Tested 5.86 0.61 2.72 1a 23 Days 0 − 0.09 0.45 0.31 2− 0.08 0.45 0.48 17 + 0.07 20.06 1.36 19 + 0.09 45.84 2.72 24 + 0.0981.03 4.57 26 + 0.07 63.30 4.15 36 + 0.31 74.78 6.90 40 + 4.03 49.535.11 1a 32 Days 0 − 0.46 0.54 0.48 22 − 0.42 0.53 0.31 24 − 0.43 0.470.32 42 + 0.46 8.79 0.99 46 + 0.44 22.26 2.11 74 + 4.22 19.82 3.99 76 +4.50 23.78 3.27

[0187] TABLE XVI Ag HCV Ag earlier HCV RNA c11- HCV Ab/Ag than Ab DayTest HCV Ab 14/c11-10 HCV Ag Dual Combo Genotype by Number Results S/COS/CO A5/c11-10 S/CO 1a 38 Days 0 − 0.09 0.49 0.53 NEG 24 + 0.11 10.636.47 POS 27 + 0.10 43.77 29.30 POS 31 + 0.11 72.92 64.97 POS 62 + 5.1944.41 37.22 POS 64 + 5.22 69.55 39.55 POS 69 + 5.91 12.92 7.54 POS 71 +6.29 7.09 4.42 POS 1b 18 Days 0 + 0.09 76.61 68.95 POS 4 + 0.09 56.0948.69 POS 7 + 0.08 39.63 57.02 POS 13 + 0.34 32.14 34.85 POS 18 + 1.5314.93 16.61 POS 21 + 3.20 19.97 13.78 POS 164 Not 5.86 0.61 0.62 NEGTested 1a 23 Days 0 − 0.09 0.45 0.80 NEG 2 − 0.08 0.45 0.43 NEG 17 +0.07 20.06 7.77 POS 19 + 0.09 45.84 17.28 POS 24 + 0.09 81.03 30.76 POS26 + 0.07 63.30 27.95 POS 36 + 0.31 74.78 36.66 POS 40 + 4.03 49.5321.78 POS 1a 32 Days 0 − 0.46 0.54 0.45 NEG 22 − 0.42 0.53 0.47 NEG 24 −0.43 0.47 0.53 NEG 42 + 0.46 8.79 5.04 POS 46 + 0.44 22.26 14.18 POS74 + 4.22 19.82 14.15 POS 76 + 4.50 23.78 8.54 POS

[0188] TABLE XVII N Tested 989* RR  9 RRR  0.91 N negative 980 Mean S/CONeg  0.12 Pop SD  0.09 SD to CO  9.76

[0189] TABLE XVIII N Tested 989* RR  1 RRR  0.10 N negative 988 MeanS/CO Neg  0.44 Pop SD  0.08 SD to CO  7.15

[0190] TABLE XIX N Tested 100* RR  0 RRR  0.00 N negative 100 Mean S/CONeg  0.56 Pop  0.15 SD to CO  2.98

[0191] TABLE XX C11-10 conjugate: C11-14 C11-3 C11-14 + C11-3microparticle microparticle Blended Panel only only microparticle PC S/N213.7 87.8 179.4 NC 78.8 90 93.25 Counts

[0192] TABLE XXI N Tested 989* RR  9 RRR  0.91 N negative 980 Mean S/CONeg  0.12 Pop SD  0.09 SD to CO  9.76

[0193] TABLE XXII N Tested 92* RR  0 RRR  0 N negative 92 Mean S/CO Neg 0.49 Pop SD  0.15 SD to CO  3.4

EXAMPLE X Binding of Monoclonal Antibodies to Three Peptides

[0194] Three new peptides were synthesized, two of which are compatiblewith an HCV Ab/Ag combo format and one suitable for use as a control.Each peptide was synthesized with a N-terminal biotin for ease oftracking during preparation of the solid phase. Peptide aa 10-53(ALAM-17) (KTKRNTNRRPQDVKFPGGGQIVGGVYLLPRRGPRLGVRATRKTS) (SEQ ID NO:37)contains HCV core amino acids 10-53 with no intervening deletions.Peptide aa 10-53{circumflex over ( )}32-50 (ALAM-18)(KTKRNTNRRPQDVKFPGGGQIVKTS) (SEQ ID NO:38) contains HCV core amino acids10-53 with a 19 amino acid deletion encompassing aa 32-50. Peptide aa10-53{circumflex over ( )}31-33{circumflex over ( )}47-48 contains HCVcore amino acids 10 through 53 where amino acids 31-33 and 47-48 weredeleted (ALAM16)(KTKRNTNRRPQDVKFPGGGQIVYLLPRRGPRLGVTRKTS) (SEQ IDNO:35). Peptides ALAM-16 and ALAM-18 are both compatible with an HCVAb/Ag combo format. In the case of ALAM-16, the deletion of amino acids31-33 prevents monoclonal c11-10 (epitope 32-36) from binding to theantigen, and the deletion of amino acids 47 and 48 prevents binding ofthe c11-14 monoclonal (epitope 45-50). ALAM-18 contains a deletion thatencompasses both the c11-10 and c11-14 binding regions. Data showing thelack of binding to ALAM-16 and ALAM-18 by monoclonals c11-10 and c11-14are shown in the following table: TABLE XXIII ALAM-18 ALAM-16 ALAM-17Peptide aa Peptide Peptide aa 10-53{circumflex over ( )}32- aa 10-53{circumflex over ( )}31- Epitope 10-53 50 33{circumflex over ( )}47-48Monoclonal (aa) S/N S/N S/N C11-15 19-27 >125.0 >200.0 >153.8 C11-1032-36 >125.0 0.9 0.4 C11-14 45-50 >125.0 0.4 0.4 C11-3 104-110 1.0 0.40.4 C11-7 112-124 0.9 0.5 0.4

[0195] In addition, 254 HCV genotyped seropositive specimens,representing HCV genotypes 1, 2, 3, 4 and 6, were tested by peptide aa10-53{circumflex over ( )}31-33{circumflex over ( )}47-48 (ALAM-16) todetermine feasibility of this peptide as an antigenic target in an HCVAb/Ag assay format. All 254 (100%) specimens were reactive toward thispeptide. Thus, the deletions present in this peptide, which serve toeliminate binding by the monoclonals necessary for Ag detection, do notnegatively impact reactivity of antibodies toward the remaining coreepitopes.

1 63 1 40 DNA Artificial Sequence Primer 1 atagaattcc atgcagaaaaaaaacaaacg taacaccaac 40 2 46 DNA Artificial Sequence Primer 2cggctgagaa cgttcagagg ttttaacgat ctgaccacca cccggg 46 3 24 DNAArtificial Sequence Primer 3 aaaacctctg aacgttctca gccg 24 4 36 DNAArtificial Sequence Primer 4 tatggatcct tattacggag acagcagcca accagc 365 244 DNA Hepatitis C Virus 5 gaattccatg cagaaaaaaa acaaacgtaacaccaaccgt cgtccgcagg acgttaaatt 60 cccgggtggt ggtcagatcg ttaaaacctctgaacgttct cagccgcgtg ggcgtcgtca 120 gccgatcccg aaagctcgtc gtccggaaggtcgtacctgg gctcagccgg gttacccgtg 180 gccgctgtac ggtaacgaag gttgcggttgggcaggttgg ctgctgtctc cgtaataagg 240 atcc 244 6 75 PRT Hepatitis C Virus6 Met Gln Lys Lys Asn Lys Arg Asn Thr Asn Arg Arg Pro Gln Asp Val 1 5 1015 Lys Phe Pro Gly Gly Gly Gln Ile Val Lys Thr Ser Glu Arg Ser Gln 20 2530 Pro Arg Gly Arg Arg Gln Pro Ile Pro Lys Ala Arg Arg Pro Glu Gly 35 4045 Arg Thr Trp Ala Gln Pro Gly Tyr Pro Trp Pro Leu Tyr Gly Asn Glu 50 5560 Gly Cys Gly Trp Ala Gly Trp Leu Leu Ser Pro 65 70 75 7 280 DNAArtificial Sequence Variant - HVC-Core Recombinant 7 gaattccatgcagaaaaaaa acaaacgtaa caccaaccgt cgtccgcagg acgttaaatt 60 cccgggtggtggtcagatcg ttggtctgct gccgcgtcgt ggtccgcgtc tgggtcgtaa 120 aacctctgaacgttctcagc cgcgtgggcg tcgtcagccg atcccgaaag ctcgtcgtcc 180 ggaaggtcgtacctgggctc agccgggtta cccgtggccg ctgtacggta acgaaggttg 240 cggttgggctggttggctgc tgtctccgta ataaggatcc 280 8 87 PRT Artificial SequenceVariant - HVC-Core Recombinant 8 Met Gln Lys Lys Asn Lys Arg Asn Thr AsnArg Arg Pro Gln Asp Val 1 5 10 15 Lys Phe Pro Gly Gly Gly Gln Ile ValGly Leu Leu Pro Arg Arg Gly 20 25 30 Pro Arg Leu Gly Arg Lys Thr Ser GluArg Ser Gln Pro Arg Gly Arg 35 40 45 Arg Gln Pro Ile Pro Lys Ala Arg ArgPro Glu Gly Arg Thr Trp Ala 50 55 60 Gln Pro Gly Tyr Pro Trp Pro Leu TyrGly Asn Glu Gly Cys Gly Trp 65 70 75 80 Ala Gly Trp Leu Leu Ser Pro 85 952 DNA Artificial Sequence Primer 9 acccagacgc ggaccacgac gcggcagcagaccaacgatc tgaccaccac cc 52 10 48 DNA Artificial Sequence Primer 10ccgcgtcgtg gtccgcgtct gggtcgtaaa acctctgaac gttctcag 48 11 301 DNAArtificial Sequence Variant - HVC-Core Recombinant 11 gaattccatgcagaaaaaaa acaaacgtaa caccaaccgt cgtccgcagg acgttaaatt 60 cccgggtggtggtcagatcg ttggtggtgt ttacgttctg ccgcgtcgtg gtccgcgtct 120 gggtgttctggctacgcgta aaacctctga acgttctcag ccgcgtgggc gtcgtcagcc 180 gatcccgaaagctcgtcgtc cggaaggtcg tacctgggct cagccgggtt acccgtggcc 240 gctgtacggtaacgaaggtt gcggttgggc tggttggctg ctgtctccgt aataaggatc 300 c 301 12 94PRT Artificial Sequence Variant - HVC-Core Recombinant 12 Met Gln LysLys Asn Lys Arg Asn Thr Asn Arg Arg Pro Gln Asp Val 1 5 10 15 Lys PhePro Gly Gly Gly Gln Ile Val Gly Gly Val Tyr Val Leu Pro 20 25 30 Arg ArgGly Pro Arg Leu Gly Val Leu Ala Thr Arg Lys Thr Ser Glu 35 40 45 Arg SerGln Pro Arg Gly Arg Arg Gln Pro Ile Pro Lys Ala Arg Arg 50 55 60 Pro GluGly Arg Thr Trp Ala Gln Pro Gly Tyr Pro Trp Pro Leu Tyr 65 70 75 80 GlyAsn Glu Gly Cys Gly Trp Ala Gly Trp Leu Leu Ser Pro 85 90 13 45 DNAArtificial Sequence Primer 13 acccagacgc ggaccacgac gcggcagaacgtaaacacca ccaac 45 14 45 DNA Artificial Sequence Primer 14 ccgcgtcgtggtccgcgtct gggtgttctg gctacgcgta aaacc 45 15 300 DNA Artificial SequenceVariant - HCV-Core Recombinant 15 gaattccatg cagaaaaaaa acaaacgtaacaccaaccgt cgtccgcagg acgttaaatt 60 cccgggtggt ggtcagatcg ttggtggtgtttacctgctn ccgcgtcgtg gtccgcgtct 120 gggtgttcgt gctacgcgta aaacctctgaacgttctcag ccgcgtgggc gtcgtcagcc 180 gatccgaaag ctcgtcgtcc ggaaggtcgtacctgggctc agccgggtta cccgtggccg 240 ctgtacggta acgaaggttg cggttgggctggttggctgc tgtctccgta ataaggatcc 300 16 94 PRT Artificial SequenceVariant - HCV-Core Recombinant 16 Met Gln Lys Lys Asn Lys Arg Asn ThrAsn Arg Arg Pro Gln Asp Val 1 5 10 15 Lys Phe Pro Gly Gly Gly Gln IleVal Gly Gly Val Tyr Leu Leu Pro 20 25 30 Arg Arg Gly Pro Arg Leu Gly ValArg Ala Thr Arg Lys Thr Ser Glu 35 40 45 Arg Ser Gln Pro Arg Gly Arg ArgGln Pro Ile Pro Lys Ala Arg Arg 50 55 60 Pro Glu Gly Arg Thr Trp Ala GlnPro Gly Tyr Pro Trp Pro Leu Tyr 65 70 75 80 Gly Asn Glu Gly Cys Gly TrpAla Gly Trp Leu Leu Ser Pro 85 90 17 1069 DNA Hepatitis C Virus p9MB-1817 gaattccatg gctgttgact ttatcccggt tgaaaatctc gagactacta tgcgttctcc 60ggttttcact gacaactctt ctccgccggt tgttccgcag tctttccagg ttgctcacct 120gcatgctccg actggttctg gtaaatctac taaagttcca gctgcttacg ctgctcaggg 180ttacaaagtt ctggttctga acccgtctgt tgctgctact ctgggtttcg gcgcctacat 240gtctaaagct cacggtatcg acccgaacat tcgtactggt gtacgtacta tcactactgg 300ttctccgatc acttactcta cttacggtaa attcctggct gacggtggtt gctctggtgg 360tgcttacgat atcatcatct gcgacgaatg ccactctact gacgctactt ctatcctggg 420tatcggtacc gttctggacc aggctgaaac tgcaggtgct cgtctggttg ttctggctac 480tgctactccg ccgggttctg ttactgttcc gcacccgaac atcgaagaag ttgctctgtc 540gactactggt gaaatcccgt tctacggtaa agctatcccg ctcgaggtta tcaaaggtgg 600tcgtcacctg attttctgcc actctaaaaa aaaatgcgac gaactggctg ctaagcttgt 660tgctctgggt atcaacgctg ttgcttacta ccgtggtctg gacgtttctg ttatcccgac 720ttctggtgac gttgttgttg tggccactga cgctctgatg actggttaca ctggtgactt 780cgactctgtt atcgattgca acacttgcaa ttccatgtct accaacccga aaccgcagaa 840aaaaaacaaa cgtaacacca accgtcgtcc gcaggacgtt aaattcccgg gtggtggtca 900gatcgttaaa acctctgaac gttctcagcc gcgtgggcgt cgtcagccga tcccgaaagc 960tcgtcgtccg gaaggtcgta cctgggctca gccgggttac ccgtggccgc tgtacggtaa 1020cgaaggttgc ggttgggctg gttggctgct gtctccgtaa taaggatcc 1069 18 350 PRTHepatitis C Virus p9MB-18 18 Met Ala Val Asp Phe Ile Pro Val Glu Asn LeuGlu Thr Thr Met Arg 1 5 10 15 Ser Pro Val Phe Thr Asp Asn Ser Ser ProPro Val Val Pro Gln Ser 20 25 30 Phe Gln Val Ala His Leu His Ala Pro ThrGly Ser Gly Lys Ser Thr 35 40 45 Lys Val Pro Ala Ala Tyr Ala Ala Gln GlyTyr Lys Val Leu Val Leu 50 55 60 Asn Pro Ser Val Ala Ala Thr Leu Gly PheGly Ala Tyr Met Ser Lys 65 70 75 80 Ala His Gly Ile Asp Pro Asn Ile ArgThr Gly Val Arg Thr Ile Thr 85 90 95 Thr Gly Ser Pro Ile Thr Tyr Ser ThrTyr Gly Lys Phe Leu Ala Asp 100 105 110 Gly Gly Cys Ser Gly Gly Ala TyrAsp Ile Ile Ile Cys Asp Glu Cys 115 120 125 His Ser Thr Asp Ala Thr SerIle Leu Gly Ile Gly Thr Val Leu Asp 130 135 140 Gln Ala Glu Thr Ala GlyAla Arg Leu Val Val Leu Ala Thr Ala Thr 145 150 155 160 Pro Pro Gly SerVal Thr Val Pro His Pro Asn Ile Glu Glu Val Ala 165 170 175 Leu Ser ThrThr Gly Glu Ile Pro Phe Tyr Gly Lys Ala Ile Pro Leu 180 185 190 Glu ValIle Lys Gly Gly Arg His Leu Ile Phe Cys His Ser Lys Lys 195 200 205 LysCys Asp Glu Leu Ala Ala Lys Leu Val Ala Leu Gly Ile Asn Ala 210 215 220Val Ala Tyr Tyr Arg Gly Leu Asp Val Ser Val Ile Pro Thr Ser Gly 225 230235 240 Asp Val Val Val Val Ala Thr Asp Ala Leu Met Thr Gly Tyr Thr Gly245 250 255 Asp Phe Asp Ser Val Ile Asp Cys Asn Thr Cys Asn Ser Met SerThr 260 265 270 Asn Pro Lys Pro Gln Lys Lys Asn Lys Arg Asn Thr Asn ArgArg Pro 275 280 285 Gln Asp Val Lys Phe Pro Gly Gly Gly Gln Ile Val LysThr Ser Glu 290 295 300 Arg Ser Gln Pro Arg Gly Arg Arg Gln Pro Ile ProLys Ala Arg Arg 305 310 315 320 Pro Glu Gly Arg Thr Trp Ala Gln Pro GlyTyr Pro Trp Pro Leu Tyr 325 330 335 Gly Asn Glu Gly Cys Gly Trp Ala GlyTrp Leu Leu Ser Pro 340 345 350 19 1051 DNA Hepatitis C Virus p9MB-19 19gaattccatg gctgttgact ttatcccggt tgaaaatctc gagactacta tgcgttctcc 60ggttttcact gacaactctt ctccgccggt tgttccgcag tctttccagg ttgctcacct 120gcatgctccg actggttctg gtaaatctac taaagttcca gctgcttacg ctgctcaggg 180ttacaaagtt ctggttctga acccgtctgt tgctgctact ctgggtttcg gcgcctacat 240gtctaaagct cacggtatcg acccgaacat tcgtactggt gtacgtacta tcactactgg 300ttctccgatc acttactcta cttacggtaa attcctggct gacggtggtt gctctggtgg 360tgcttacgat atcatcatct gcgacgaatg ccactctact gacgctactt ctatcctggg 420tatcggtacc gttctggacc aggctgaaac tgcaggtgct cgtctggttg ttctggctac 480tgctactccg ccgggttctg ttactgttcc gcacccgaac atcgaagaag ttgctctgtc 540gactactggt gaaatcccgt tctacggtaa agctatcccg ctcgaggtta tcaaaggtgg 600tcgtcacctg attttctgcc actctaaaaa aaaatgcgac gaactggctg ctaagcttgt 660tgctctgggt atcaacgctg ttgcttacta ccgtggtctg gacgtttctg ttatcccgac 720ttctggtgac gttgttgttg tggccactga cgctctgatg actggttaca ctggtgactt 780cgactctgtt atcgattgca acacttgcaa ttccatgcag aaaaaaaaca aacgtaacac 840caaccgtcgt ccgcaggacg ttaaattccc gggtggtggt cagatcgtta aaacctctga 900acgttctcag ccgcgtgggc gtcgtcagcc gatcccgaaa gctcgtcgtc cggaaggtcg 960tacctgggct cagccgggtt acccgtggcc gctgtacggt aacgaaggtt gcggttgggc 1020tggttggctg ctgtctccgt aataaggatc c 1051 20 344 PRT Hepatitis C Virusp9MB-19 20 Met Ala Val Asp Phe Ile Pro Val Glu Asn Leu Glu Thr Thr MetArg 1 5 10 15 Ser Pro Val Phe Thr Asp Asn Ser Ser Pro Pro Val Val ProGln Ser 20 25 30 Phe Gln Val Ala His Leu His Ala Pro Thr Gly Ser Gly LysSer Thr 35 40 45 Lys Val Pro Ala Ala Tyr Ala Ala Gln Gly Tyr Lys Val LeuVal Leu 50 55 60 Asn Pro Ser Val Ala Ala Thr Leu Gly Phe Gly Ala Tyr MetSer Lys 65 70 75 80 Ala His Gly Ile Asp Pro Asn Ile Arg Thr Gly Val ArgThr Ile Thr 85 90 95 Thr Gly Ser Pro Ile Thr Tyr Ser Thr Tyr Gly Lys PheLeu Ala Asp 100 105 110 Gly Gly Cys Ser Gly Gly Ala Tyr Asp Ile Ile IleCys Asp Glu Cys 115 120 125 His Ser Thr Asp Ala Thr Ser Ile Leu Gly IleGly Thr Val Leu Asp 130 135 140 Gln Ala Glu Thr Ala Gly Ala Arg Leu ValVal Leu Ala Thr Ala Thr 145 150 155 160 Pro Pro Gly Ser Val Thr Val ProHis Pro Asn Ile Glu Glu Val Ala 165 170 175 Leu Ser Thr Thr Gly Glu IlePro Phe Tyr Gly Lys Ala Ile Pro Leu 180 185 190 Glu Val Ile Lys Gly GlyArg His Leu Ile Phe Cys His Ser Lys Lys 195 200 205 Lys Cys Asp Glu LeuAla Ala Lys Leu Val Ala Leu Gly Ile Asn Ala 210 215 220 Val Ala Tyr TyrArg Gly Leu Asp Val Ser Val Ile Pro Thr Ser Gly 225 230 235 240 Asp ValVal Val Val Ala Thr Asp Ala Leu Met Thr Gly Tyr Thr Gly 245 250 255 AspPhe Asp Ser Val Ile Asp Cys Asn Thr Cys Asn Ser Met Gln Lys 260 265 270Lys Asn Lys Arg Asn Thr Asn Arg Arg Pro Gln Asp Val Lys Phe Pro 275 280285 Gly Gly Gly Gln Ile Val Lys Thr Ser Glu Arg Ser Gln Pro Arg Gly 290295 300 Arg Arg Gln Pro Ile Pro Lys Ala Arg Arg Pro Glu Gly Arg Thr Trp305 310 315 320 Ala Gln Pro Gly Tyr Pro Trp Pro Leu Tyr Gly Asn Glu GlyCys Gly 325 330 335 Trp Ala Gly Trp Leu Leu Ser Pro 340 21 30 DNAArtificial Sequence Primer 21 tatagaattc catggctgtt gactttatcc 30 22 27DNA Artificial Sequence Primer 22 ggaattgcaa gtgttgcaat cgataac 27 23 51DNA Artificial Sequence Primer 23 gttatcgatt gcaacacttg caattccatgcagaaaaaaa acaaacgtaa c 51 24 1066 DNA Hepatitis C Virus p9MB-20 24gaattccatg gctgttgact ttatcccggt tgaaaatctc gagactacta tgcgttctcc 60ggttttcact gacaactctt ctccgccggt tgttccgcag tctttccagg ttgctcacct 120gcatgctccg actggttctg gtaaatctac taaagttcca gctgcttacg ctgctcaggg 180ttacaaagtt ctggttctga acccgtctgt tgctgctact ctgggtttcg gcgcctacat 240gtctaaagct cacggtatcg acccgaacat tcgtactggt gtacgtacta tcactactgg 300ttctccgatc acttactcta cttacggtaa attcctggct gacggtggtt gctctggtgg 360tgcttacgat atcatcatct gcgacgaatg ccactctact gacgctactt ctatcctggg 420tatcggtacc gttctggacc aggctgaaac tgcaggtgct cgtctggttg ttctggctac 480tgctactccg ccgggttctg ttactgttcc gcacccgaac atcgaagaag ttgctctgtc 540gactactggt gaaatcccgt tctacggtaa agctatcccg ctcgaggtta tcaaaggtgg 600tcgtcacctg attttctgcc actctaaaaa aaaatgcgac gaactggctg ctaagcttgt 660tgctctgggt atcaacgctg ttgcttacta ccgtggtctg gacgtttctg ttatcccgac 720ttctggtgac gttgttgttg tggccactga cgctctgatg actggttaca ctggtgactt 780cgactctgtt atcgattgca acacttgcaa ttccggtggt ggtggttcta tgcagaaaaa 840aaacaaacgt aacaccaacc gtcgtccgca ggacgttaaa ttcccgggtg gtggtcagat 900cgttaaaacc tctgaacgtt ctcagccgcg tgggcgtcgt cagccgatcc cgaaagctcg 960tcgtccggaa ggtcgtacct gggctcagcc gggttacccg tggccgctgt acggtaacga 1020aggttgcggt tgggctggtt ggctgctgtc tccgtaataa ggatcc 1066 25 349 PRTHepatitis C Virus p9MB-20 25 Met Ala Val Asp Phe Ile Pro Val Glu Asn LeuGlu Thr Thr Met Arg 1 5 10 15 Ser Pro Val Phe Thr Asp Asn Ser Ser ProPro Val Val Pro Gln Ser 20 25 30 Phe Gln Val Ala His Leu His Ala Pro ThrGly Ser Gly Lys Ser Thr 35 40 45 Lys Val Pro Ala Ala Tyr Ala Ala Gln GlyTyr Lys Val Leu Val Leu 50 55 60 Asn Pro Ser Val Ala Ala Thr Leu Gly PheGly Ala Tyr Met Ser Lys 65 70 75 80 Ala His Gly Ile Asp Pro Asn Ile ArgThr Gly Val Arg Thr Ile Thr 85 90 95 Thr Gly Ser Pro Ile Thr Tyr Ser ThrTyr Gly Lys Phe Leu Ala Asp 100 105 110 Gly Gly Cys Ser Gly Gly Ala TyrAsp Ile Ile Ile Cys Asp Glu Cys 115 120 125 His Ser Thr Asp Ala Thr SerIle Leu Gly Ile Gly Thr Val Leu Asp 130 135 140 Gln Ala Glu Thr Ala GlyAla Arg Leu Val Val Leu Ala Thr Ala Thr 145 150 155 160 Pro Pro Gly SerVal Thr Val Pro His Pro Asn Ile Glu Glu Val Ala 165 170 175 Leu Ser ThrThr Gly Glu Ile Pro Phe Tyr Gly Lys Ala Ile Pro Leu 180 185 190 Glu ValIle Lys Gly Gly Arg His Leu Ile Phe Cys His Ser Lys Lys 195 200 205 LysCys Asp Glu Leu Ala Ala Lys Leu Val Ala Leu Gly Ile Asn Ala 210 215 220Val Ala Tyr Tyr Arg Gly Leu Asp Val Ser Val Ile Pro Thr Ser Gly 225 230235 240 Asp Val Val Val Val Ala Thr Asp Ala Leu Met Thr Gly Tyr Thr Gly245 250 255 Asp Phe Asp Ser Val Ile Asp Cys Asn Thr Cys Asn Ser Gly GlyGly 260 265 270 Gly Ser Met Gln Lys Lys Asn Lys Arg Asn Thr Asn Arg ArgPro Gln 275 280 285 Asp Val Lys Phe Pro Gly Gly Gly Gln Ile Val Lys ThrSer Glu Arg 290 295 300 Ser Gln Pro Arg Gly Arg Arg Gln Pro Ile Pro LysAla Arg Arg Pro 305 310 315 320 Glu Gly Arg Thr Trp Ala Gln Pro Gly TyrPro Trp Pro Leu Tyr Gly 325 330 335 Asn Glu Gly Cys Gly Trp Ala Gly TrpLeu Leu Ser Pro 340 345 26 66 DNA Artificial Sequence Primer 26gttatcgatt gcaacacttg caattccggt ggtggtggtt ctatgcagaa aaaaaacaaa 60cgtaac 66 27 1293 DNA Hepatitis C Virus p9MB-22 27 gaattccatg gctgttgactttatcccggt tgaaaatctc gagactacta tgcgttctcc 60 ggttttcact gacaactcttctccgccggt tgttccgcag tctttccagg ttgctcacct 120 gcatgctccg actggttctggtaaatctac taaagttcca gctgcttacg ctgctcaggg 180 ttacaaagtt ctggttctgaacccgtctgt tgctgctact ctgggtttcg gcgcctacat 240 gtctaaagct cacggtatcgacccgaacat tcgtactggt gtacgtacta tcactactgg 300 ttctccgatc acttactctacttacggtaa attcctggct gacggtggtt gctctggtgg 360 tgcttacgat atcatcatctgcgacgaatg ccactctact gacgctactt ctatcctggg 420 tatcggtacc gttctggaccaggctgaaac tgcaggtgct cgtctggttg ttctggctac 480 tgctactccg ccgggttctgttactgttcc gcacccgaac atcgaagaag ttgctctgtc 540 gactactggt gaaatcccgttctacggtaa agctatcccg ctcgaggtta tcaaaggtgg 600 tcgtcacctg attttctgccactctaaaaa aaaatgcgac gaactggctg ctaagcttgt 660 tgctctgggt atcaacgctgttgcttacta ccgtggtctg gacgtttctg ttatcccgac 720 ttctggtgac gttgttgttgtggccactga cgctctgatg actggttaca ctggtgactt 780 cgactctgtt atcgattgcaacacttgcaa ttccggtggt ggtggttcta tgtctaccaa 840 cccgaaaccg cagaaaaaaaacaaacgtaa caccaaccgt cgtccgcagg acgttaaatt 900 cccgggtggt ggtcagatcgttggtggtgt ttacctgctg ccgcgtcgtg gtccgcgtct 960 gggtgttcgt gctacgcgtaaaacctctga acgttctcag ccgcgtgggc gtcgtcagcc 1020 gatcccgaaa gctcgtcgtccggaaggtcg tacctgggct cagccgggtt acccgtggcc 1080 gctgtacggt aacgaaggttgcggttgggc tggttggctg ctgtctccgc gtggatctcg 1140 tccgtcttgg ggtccgaccgacccgcgtcg tcgttctcgt aaccttggta aagttatcga 1200 taccctgacc tgcggtttcgctgacctgat gggttacata ccgctggttg gagctccgct 1260 gggtggtgct gctcgtgcttaacccatgga tcc 1293 28 424 PRT Hepatitis C Virus p9MB-22 28 Met Ala ValAsp Phe Ile Pro Val Glu Asn Leu Glu Thr Thr Met Arg 1 5 10 15 Ser ProVal Phe Thr Asp Asn Ser Ser Pro Pro Val Val Pro Gln Ser 20 25 30 Phe GlnVal Ala His Leu His Ala Pro Thr Gly Ser Gly Lys Ser Thr 35 40 45 Lys ValPro Ala Ala Tyr Ala Ala Gln Gly Tyr Lys Val Leu Val Leu 50 55 60 Asn ProSer Val Ala Ala Thr Leu Gly Phe Gly Ala Tyr Met Ser Lys 65 70 75 80 AlaHis Gly Ile Asp Pro Asn Ile Arg Thr Gly Val Arg Thr Ile Thr 85 90 95 ThrGly Ser Pro Ile Thr Tyr Ser Thr Tyr Gly Lys Phe Leu Ala Asp 100 105 110Gly Gly Cys Ser Gly Gly Ala Tyr Asp Ile Ile Ile Cys Asp Glu Cys 115 120125 His Ser Thr Asp Ala Thr Ser Ile Leu Gly Ile Gly Thr Val Leu Asp 130135 140 Gln Ala Glu Thr Ala Gly Ala Arg Leu Val Val Leu Ala Thr Ala Thr145 150 155 160 Pro Pro Gly Ser Val Thr Val Pro His Pro Asn Ile Glu GluVal Ala 165 170 175 Leu Ser Thr Thr Gly Glu Ile Pro Phe Tyr Gly Lys AlaIle Pro Leu 180 185 190 Glu Val Ile Lys Gly Gly Arg His Leu Ile Phe CysHis Ser Lys Lys 195 200 205 Lys Cys Asp Glu Leu Ala Ala Lys Leu Val AlaLeu Gly Ile Asn Ala 210 215 220 Val Ala Tyr Tyr Arg Gly Leu Asp Val SerVal Ile Pro Thr Ser Gly 225 230 235 240 Asp Val Val Val Val Ala Thr AspAla Leu Met Thr Gly Tyr Thr Gly 245 250 255 Asp Phe Asp Ser Val Ile AspCys Asn Thr Cys Asn Ser Gly Gly Gly 260 265 270 Gly Ser Met Ser Thr AsnPro Lys Pro Gln Lys Lys Asn Lys Arg Asn 275 280 285 Thr Asn Arg Arg ProGln Asp Val Lys Phe Pro Gly Gly Gly Gln Ile 290 295 300 Val Gly Gly ValTyr Leu Leu Pro Arg Arg Gly Pro Arg Leu Gly Val 305 310 315 320 Arg AlaThr Arg Lys Thr Ser Glu Arg Ser Gln Pro Arg Gly Arg Arg 325 330 335 GlnPro Ile Pro Lys Ala Arg Arg Pro Glu Gly Arg Thr Trp Ala Gln 340 345 350Pro Gly Tyr Pro Trp Pro Leu Tyr Gly Asn Glu Gly Cys Gly Trp Ala 355 360365 Gly Trp Leu Leu Ser Pro Arg Gly Ser Arg Pro Ser Trp Gly Pro Thr 370375 380 Asp Pro Arg Arg Arg Ser Arg Asn Leu Gly Lys Val Ile Asp Thr Leu385 390 395 400 Thr Cys Gly Phe Ala Asp Leu Met Gly Tyr Ile Pro Leu ValGly Ala 405 410 415 Pro Leu Gly Gly Ala Ala Arg Ala 420 29 66 DNAArtificial Sequence Primer 29 gttatcgatt gcaacacttg caattccggtggtggtggtt ctatgtctac caacccgaaa 60 ccgcag 66 30 27 DNA ArtificialSequence Primer 30 tataggatcc atgggttaag cacgagc 27 31 1111 DNAHepatitis C Virus p9MB-31 31 gaattccatg gctgttgact ttatcccggt tgaaaatctcgagactacta tgcgttctcc 60 ggttttcact gacaactctt ctccgccggt tgttccgcagtctttccagg ttgctcacct 120 gcatgctccg actggttctg gtaaatctac taaagttccagctgcttacg ctgctcaggg 180 ttacaaagtt ctggttctga acccgtctgt tgctgctactctgggtttcg gcgcctacat 240 gtctaaagct cacggtatcg acccgaacat tcgtactggtgtacgtacta tcactactgg 300 ttctccgatc acttactcta cttacggtaa attcctggctgacggtggtt gctctggtgg 360 tgcttacgat atcatcatct gcgacgaatg ccactctactgacgctactt ctatcctggg 420 tatcggtacc gttctggacc aggctgaaac tgcaggtgctcgtctggttg ttctggctac 480 tgctactccg ccgggttctg ttactgttcc gcacccgaacatcgaagaag ttgctctgtc 540 gactactggt gaaatcccgt tctacggtaa agctatcccgctcgaggtta tcaaaggtgg 600 tcgtcacctg attttctgcc actctaaaaa aaaatgcgacgaactggctg ctaagcttgt 660 tgctctgggt atcaacgctg ttgcttacta ccgtggtctggacgtttctg ttatcccgac 720 ttctggtgac gttgttgttg tggccactga cgctctgatgactggttaca ctggtgactt 780 cgactctgtt atcgattgca acacttgcaa ttccatgtctaccaacccga aaccgcagaa 840 aaaaaacaaa cgtaacacca accgtcgtcc gcaggacgttaaattcccgg gtggtggtca 900 gatcgtttac ctgctgccgc gtcgtggtcc gcgtctgggtgttacgcgta aaacctctga 960 acgttctcag ccgcgtgggc gtcgtcagcc gatcccgaaagctcgtcgtc cggaaggtcg 1020 tacctgggct cagccgggtt acccgtggcc gctgtacggtaacgaaggtt gcggttgggc 1080 tggttggcta ctgtctccgt aataaggatc c 1111 32364 PRT Hepatitis C Virus p9MB-31 32 Met Ala Val Asp Phe Ile Pro Val GluAsn Leu Glu Thr Thr Met Arg 1 5 10 15 Ser Pro Val Phe Thr Asp Asn SerSer Pro Pro Val Val Pro Gln Ser 20 25 30 Phe Gln Val Ala His Leu His AlaPro Thr Gly Ser Gly Lys Ser Thr 35 40 45 Lys Val Pro Ala Ala Tyr Ala AlaGln Gly Tyr Lys Val Leu Val Leu 50 55 60 Asn Pro Ser Val Ala Ala Thr LeuGly Phe Gly Ala Tyr Met Ser Lys 65 70 75 80 Ala His Gly Ile Asp Pro AsnIle Arg Thr Gly Val Arg Thr Ile Thr 85 90 95 Thr Gly Ser Pro Ile Thr TyrSer Thr Tyr Gly Lys Phe Leu Ala Asp 100 105 110 Gly Gly Cys Ser Gly GlyAla Tyr Asp Ile Ile Ile Cys Asp Glu Cys 115 120 125 His Ser Thr Asp AlaThr Ser Ile Leu Gly Ile Gly Thr Val Leu Asp 130 135 140 Gln Ala Glu ThrAla Gly Ala Arg Leu Val Val Leu Ala Thr Ala Thr 145 150 155 160 Pro ProGly Ser Val Thr Val Pro His Pro Asn Ile Glu Glu Val Ala 165 170 175 LeuSer Thr Thr Gly Glu Ile Pro Phe Tyr Gly Lys Ala Ile Pro Leu 180 185 190Glu Val Ile Lys Gly Gly Arg His Leu Ile Phe Cys His Ser Lys Lys 195 200205 Lys Cys Asp Glu Leu Ala Ala Lys Leu Val Ala Leu Gly Ile Asn Ala 210215 220 Val Ala Tyr Tyr Arg Gly Leu Asp Val Ser Val Ile Pro Thr Ser Gly225 230 235 240 Asp Val Val Val Val Ala Thr Asp Ala Leu Met Thr Gly TyrThr Gly 245 250 255 Asp Phe Asp Ser Val Ile Asp Cys Asn Thr Cys Asn SerMet Ser Thr 260 265 270 Asn Pro Lys Pro Gln Lys Lys Asn Lys Arg Asn ThrAsn Arg Arg Pro 275 280 285 Gln Asp Val Lys Phe Pro Gly Gly Gly Gln IleVal Tyr Leu Leu Pro 290 295 300 Arg Arg Gly Pro Arg Leu Gly Val Thr ArgLys Thr Ser Glu Arg Ser 305 310 315 320 Gln Pro Arg Gly Arg Arg Gln ProIle Pro Lys Ala Arg Arg Pro Glu 325 330 335 Gly Arg Thr Trp Ala Gln ProGly Tyr Pro Trp Pro Leu Tyr Gly Asn 340 345 350 Glu Gly Cys Gly Trp AlaGly Trp Leu Leu Ser Pro 355 360 33 52 DNA Artificial Sequence Primer 33acccagacgc ggaccacgac gcggcagcag gtaaacgatc tgaccaccac cc 52 34 54 DNAArtificial Sequence Primer 34 ccgcgtcgtg gtccgcgtct gggtgttacgcgtaaaacct ctgaacgttc tcag 54 35 1105 DNA Hepatitis A Virus p9MB-24 35gaattccatg gctgttgact ttatcccggt tgaaaatctc gagactacta tgcgttctcc 60ggttttcact gacaactctt ctccgccggt tgttccgcag tctttccagg ttgctcacct 120gcatgctccg actggttctg gtaaatctac taaagttcca gctgcttacg ctgctcaggg 180ttacaaagtt ctggttctga acccgtctgt tgctgctact ctgggtttcg gcgcctacat 240gtctaaagct cacggtatcg acccgaacat tcgtactggt gtacgtacta tcactactgg 300ttctccgatc acttactcta cttacggtaa attcctggct gacggtggtt gctctggtgg 360tgcttacgat atcatcatct gcgacgaatg ccactctact gacgctactt ctatcctggg 420tatcggtacc gttctggacc aggctgaaac tgcaggtgct cgtctggttg ttctggctac 480tgctactccg ccgggttctg ttactgttcc gcacccgaac atcgaagaag ttgctctgtc 540gactactggt gaaatcccgt tctacggtaa agctatcccg ctcgaggtta tcaaaggtgg 600tcgtcacctg attttctgcc actctaaaaa aaaatgcgac gaactggctg ctaagcttgt 660tgctctgggt atcaacgctg ttgcttacta ccgtggtctg gacgtttctg ttatcccgac 720ttctggtgac gttgttgttg tggccactga cgctctgatg actggttaca ctggtgactt 780cgactctgtt atcgattgca acacttgcaa ttccatgtct accaacccga aaccgcagaa 840aaaaaacaaa cgtaacacca accgtcgtcc gcaggacgtt aaattcccgg gtggtggtca 900gatcgttggt ctgctgccgc gtcgtggtcc gcgtctgggt cgtaaaacct ctgaacgttc 960tcagccgcgt gggcgtcgtc agccgatccc gaaagctcgt cgtccggaag gtcgtacctg 1020ggctcagccg ggttacccgt ggccgctgta cggtaacgaa ggttgcggtt gggctggttg 1080gctgctgtct ccgtaataag gatcc 1105 36 358 PRT Hepatitis C Virus p9MB-24 36Met Ala Val Asp Phe Ile Pro Val Glu Asn Leu Glu Thr Thr Met Arg 1 5 1015 Ser Pro Val Phe Thr Asp Asn Ser Ser Pro Pro Val Val Pro Gln Ser 20 2530 Phe Gln Val Ala His Leu His Ala Pro Thr Gly Ser Gly Lys Ser Thr 35 4045 Lys Val Pro Ala Ala Tyr Ala Ala Gln Gly Tyr Lys Val Leu Val Leu 50 5560 Asn Pro Ser Val Ala Ala Thr Leu Gly Phe Gly Ala Tyr Met Ser Lys 65 7075 80 Ala His Gly Ile Asp Pro Asn Ile Arg Thr Gly Val Arg Thr Ile Thr 8590 95 Thr Gly Ser Pro Ile Thr Tyr Ser Thr Tyr Gly Lys Phe Leu Ala Asp100 105 110 Gly Gly Cys Ser Gly Gly Ala Tyr Asp Ile Ile Ile Cys Asp GluCys 115 120 125 His Ser Thr Asp Ala Thr Ser Ile Leu Gly Ile Gly Thr ValLeu Asp 130 135 140 Gln Ala Glu Thr Ala Gly Ala Arg Leu Val Val Leu AlaThr Ala Thr 145 150 155 160 Pro Pro Gly Ser Val Thr Val Pro His Pro AsnIle Glu Glu Val Ala 165 170 175 Leu Ser Thr Thr Gly Glu Ile Pro Phe TyrGly Lys Ala Ile Pro Leu 180 185 190 Glu Val Ile Lys Gly Gly Arg His LeuIle Phe Cys His Ser Lys Lys 195 200 205 Lys Cys Asp Glu Leu Ala Ala LysLeu Val Ala Leu Gly Ile Asn Ala 210 215 220 Val Ala Tyr Tyr Arg Gly LeuAsp Val Ser Val Ile Pro Thr Ser Gly 225 230 235 240 Asp Val Val Val ValAla Thr Asp Ala Leu Met Thr Gly Tyr Thr Gly 245 250 255 Asp Phe Asp SerVal Ile Asp Cys Asn Thr Cys Asn Ser Met Ser Thr 260 265 270 Asn Pro LysPro Gln Lys Lys Asn Lys Arg Asn Thr Asn Arg Arg Pro 275 280 285 Gln AspVal Lys Phe Pro Gly Gly Gly Gln Ile Val Leu Leu Pro Arg 290 295 300 ArgGly Pro Arg Leu Gly Arg Lys Thr Ser Glu Arg Ser Gln Pro Arg 305 310 315320 Gly Arg Arg Gln Pro Ile Pro Lys Ala Arg Arg Pro Glu Gly Arg Thr 325330 335 Trp Ala Gln Pro Gly Tyr Pro Trp Pro Leu Tyr Gly Asn Gly Trp Ala340 345 350 Gly Trp Leu Leu Ser Pro 355 37 1120 DNA Hepatitis C Virusp9MB-25 37 gaattccatg gctgttgact ttatcccggt tgaaaatctc gagactactatgcgttctcc 60 ggttttcact gacaactctt ctccgccggt tgttccgcag tctttccaggttgctcacct 120 gcatgctccg actggttctg gtaaatctac taaagttcca gctgcttacgctgctcaggg 180 ttacaaagtt ctggttctga acccgtctgt tgctgctact ctgggtttcggcgcctacat 240 gtctaaagct cacggtatcg acccgaacat tcgtactggt gtacgtactatcactactgg 300 ttctccgatc acttactcta cttacggtaa attcctggct gacggtggttgctctggtgg 360 tgcttacgat atcatcatct gcgacgaatg ccactctact gacgctacttctatcctggg 420 tatcggtacc gttctggacc aggctgaaac tgcaggtgct cgtctggttgttctggctac 480 tgctactccg ccgggttctg ttactgttcc gcacccgaac atcgaagaagttgctctgtc 540 gactactggt gaaatcccgt tctacggtaa agctatcccg ctcgaggttatcaaaggtgg 600 tcgtcacctg attttctgcc actctaaaaa aaaatgcgac gaactggctgctaagcttgt 660 tgctctgggt atcaacgctg ttgcttacta ccgtggtctg gacgtttctgttatcccgac 720 ttctggtgac gttgttgttg tggccactga cgctctgatg actggttacactggtgactt 780 cgactctgtt atcgattgca acacttgcaa ttccggtggt ggtggttctatgtctaccaa 840 cccgaaaccg cagaaaaaaa acaaacgtaa caccaaccgt cgtccgcaggacgttaaatt 900 cccgggtggt ggtcagatcg ttggtctgct gccgcgtcgt ggtccgcgtctgggtcgtaa 960 aacctctgaa cgttctcagc cgcgtgggcg tcgtcagccg atcccgaaagctcgtcgtcc 1020 ggaaggtcgt acctgggctc agccgggtta cccgtggccg ctgtacggtaacgaaggttg 1080 cggttgggct ggttggctgc tgtctccgta ataaggatcc 1120 38 367PRT Hepatitis C Virus p9MB-25 38 Met Ala Val Asp Phe Ile Pro Val Glu AsnLeu Glu Thr Thr Met Arg 1 5 10 15 Ser Pro Val Phe Thr Asp Asn Ser SerPro Pro Val Val Pro Gln Ser 20 25 30 Phe Gln Val Ala His Leu His Ala ProThr Gly Ser Gly Lys Ser Thr 35 40 45 Lys Val Pro Ala Ala Tyr Ala Ala GlnGly Tyr Lys Val Leu Val Leu 50 55 60 Asn Pro Ser Val Ala Ala Thr Leu GlyPhe Gly Ala Tyr Met Ser Lys 65 70 75 80 Ala His Gly Ile Asp Pro Asn IleArg Thr Gly Val Arg Thr Ile Thr 85 90 95 Thr Gly Ser Pro Ile Thr Tyr SerThr Tyr Gly Lys Phe Leu Ala Asp 100 105 110 Gly Gly Cys Ser Gly Gly AlaTyr Asp Ile Ile Ile Cys Asp Glu Cys 115 120 125 His Ser Thr Asp Ala ThrSer Ile Leu Gly Ile Gly Thr Val Leu Asp 130 135 140 Gln Ala Glu Thr AlaGly Ala Arg Leu Val Val Leu Ala Thr Ala Thr 145 150 155 160 Pro Pro GlySer Val Thr Val Pro His Pro Asn Ile Glu Glu Val Ala 165 170 175 Leu SerThr Thr Gly Glu Ile Pro Phe Tyr Gly Lys Ala Ile Pro Leu 180 185 190 GluVal Ile Lys Gly Gly Arg His Leu Ile Phe Cys His Ser Lys Lys 195 200 205Lys Cys Asp Glu Leu Ala Ala Lys Leu Val Ala Leu Gly Ile Asn Ala 210 215220 Val Ala Tyr Tyr Arg Gly Leu Asp Val Ser Val Ile Pro Thr Ser Gly 225230 235 240 Asp Val Val Val Val Ala Thr Asp Ala Leu Met Thr Gly Tyr ThrGly 245 250 255 Asp Phe Asp Ser Val Ile Asp Cys Asn Thr Cys Asn Ser GlyGly Gly 260 265 270 Gly Ser Met Ser Thr Asn Pro Lys Pro Gln Lys Lys AsnLys Arg Asn 275 280 285 Thr Asn Arg Arg Pro Gln Asp Val Lys Phe Pro GlyGly Gly Gln Ile 290 295 300 Val Gly Leu Leu Pro Arg Arg Gly Pro Arg LeuGly Arg Lys Thr Ser 305 310 315 320 Glu Arg Ser Gln Pro Arg Gly Arg ArgGln Pro Ile Pro Lys Ala Arg 325 330 335 Arg Pro Glu Gly Arg Thr Trp AlaGln Pro Gly Tyr Pro Trp Pro Leu 340 345 350 Tyr Gly Asn Glu Gly Cys GlyTrp Ala Gly Trp Leu Leu Ser Pro 355 360 365 39 1084 DNA Hepatitis CVirus p9MB-26 39 gaattccatg gctgttgact ttatcccggt tgaaaatctc gagactactatgcgttctcc 60 ggttttcact gacaactctt ctccgccggt tgttccgcag tctttccaggttgctcacct 120 gcatgctccg actggttctg gtaaatctac taaagttcca gctgcttacgctgctcaggg 180 ttacaaagtt ctggttctga acccgtctgt tgctgctact ctgggtttcggcgcctacat 240 gtctaaagct cacggtatcg acccgaacat tcgtactggt gtacgtactatcactactgg 300 ttctccgatc acttactcta cttacggtaa attcctggct gacggtggttgctctggtgg 360 tgcttacgat atcatcatct gcgacgaatg ccactctact gacgctacttctatcctggg 420 tatcggtacc gttctggacc aggctgaaac tgcaggtgct cgtctggttgttctggctac 480 tgctactccg ccgggttctg ttactgttcc gcacccgaac atcgaagaagttgctctgtc 540 gactactggt gaaatcccgt tctacggtaa agctatcccg ctcgaggttatcaaaggtgg 600 tcgtcacctg attttctgcc actctaaaaa aaaatgcgac gaactggctgctaagcttgt 660 tgctctgggt atcaacgctg ttgcttacta ccgtggtctg gacgtttctgttatcccgac 720 ttctggtgac gttgttgttg tggccactga cgctctgatg actggttacactggtgactt 780 cgactctgtt atcgattgca acacttgcaa ttccggtggt ggtggttctatgtctaccaa 840 cccgaaaccg cagaaaaaaa acaaacgtaa caccaaccgt cgtccgcaggacgttaaatt 900 cccgggtggt ggtcagatcg ttaaaacctc tgaacgttct cagccgcgtgggcgtcgtca 960 gccgatcccg aaagctcgtc gtccggaagg tcgtacctgg gctcagccgggttacccgtg 1020 gccgctgtac ggtaacgaag gttgcggttg ggctggttgg ctgctgtctccgtaataagg 1080 atcc 1084 40 355 PRT Hepatitis C Virus p9MB-26 40 MetAla Val Asp Phe Ile Pro Val Glu Asn Leu Glu Thr Thr Met Arg 1 5 10 15Ser Pro Val Phe Thr Asp Asn Ser Ser Pro Pro Val Val Pro Gln Ser 20 25 30Phe Gln Val Ala His Leu His Ala Pro Thr Gly Ser Gly Lys Ser Thr 35 40 45Lys Val Pro Ala Ala Tyr Ala Ala Gln Gly Tyr Lys Val Leu Val Leu 50 55 60Asn Pro Ser Val Ala Ala Thr Leu Gly Phe Gly Ala Tyr Met Ser Lys 65 70 7580 Ala His Gly Ile Asp Pro Asn Ile Arg Thr Gly Val Arg Thr Ile Thr 85 9095 Thr Gly Ser Pro Ile Thr Tyr Ser Thr Tyr Gly Lys Phe Leu Ala Asp 100105 110 Gly Gly Cys Ser Gly Gly Ala Tyr Asp Ile Ile Ile Cys Asp Glu Cys115 120 125 His Ser Thr Asp Ala Thr Ser Ile Leu Gly Ile Gly Thr Val LeuAsp 130 135 140 Gln Ala Glu Thr Ala Gly Ala Arg Leu Val Val Leu Ala ThrAla Thr 145 150 155 160 Pro Pro Gly Ser Val Thr Val Pro His Pro Asn IleGlu Glu Val Ala 165 170 175 Leu Ser Thr Thr Gly Glu Ile Pro Phe Tyr GlyLys Ala Ile Pro Leu 180 185 190 Glu Val Ile Lys Gly Gly Arg His Leu IlePhe Cys His Ser Lys Lys 195 200 205 Lys Cys Asp Glu Leu Ala Ala Lys LeuVal Ala Leu Gly Ile Asn Ala 210 215 220 Val Ala Tyr Tyr Arg Gly Leu AspVal Ser Val Ile Pro Thr Ser Gly 225 230 235 240 Asp Val Val Val Val AlaThr Asp Ala Leu Met Thr Gly Tyr Thr Gly 245 250 255 Asp Phe Asp Ser ValIle Asp Cys Asn Thr Cys Asn Ser Gly Gly Gly 260 265 270 Gly Ser Met SerThr Asn Pro Lys Pro Gln Lys Lys Asn Lys Arg Asn 275 280 285 Thr Asn ArgArg Pro Gln Asp Val Lys Phe Pro Gly Gly Gly Gln Ile 290 295 300 Val LysThr Ser Glu Arg Ser Gln Pro Arg Gly Arg Arg Gln Pro Ile 305 310 315 320Pro Lys Ala Arg Arg Pro Glu Gly Arg Thr Trp Ala Gln Pro Gly Tyr 325 330335 Pro Trp Pro Leu Tyr Gly Asn Glu Gly Cys Gly Trp Ala Gly Trp Leu 340345 350 Leu Ser Pro 355 41 39 PRT Hepatitis C Virus ALAM-16 41 Lys ThrLys Arg Asn Thr Asn Arg Arg Pro Gln Asp Val Lys Phe Pro 1 5 10 15 GlyGly Gly Gln Ile Val Tyr Leu Leu Pro Arg Arg Gly Pro Arg Leu 20 25 30 GlyVal Thr Arg Lys Thr Ser 35 42 44 PRT Hepatitis C Virus ALAM-17 42 LysThr Lys Arg Asn Thr Asn Arg Arg Pro Gln Asp Val Lys Phe Pro 1 5 10 15Gly Gly Gly Gln Ile Val Gly Gly Val Tyr Leu Leu Pro Arg Arg Gly 20 25 30Pro Arg Leu Gly Val Arg Ala Thr Arg Lys Thr Ser 35 40 43 25 PRTHepatitis C Virus ALAM-18 43 Lys Thr Lys Arg Asn Thr Asn Arg Arg Pro GlnAsp Val Lys Phe Pro 1 5 10 15 Gly Gly Gly Gln Ile Val Lys Thr Ser 20 2544 18 DNA Artificial Sequence Primer 44 gatcgctcga attcctcg 18 45 20 DNAArtificial Sequence Primer 45 cgaggaattc gagcgatctt 20 46 18 PRTArtificial Sequence HCV-Core derived peptides 46 Met Ser Thr Asn Pro LysPro Gln Lys Lys Asn Lys Arg Asn Thr Asn 1 5 10 15 Arg Arg 47 18 PRTArtificial Sequence HCV-Core derived peptides 47 Asn Lys Arg Asn Thr AsnArg Arg Pro Gln Asp Val Lys Phe Pro Gly 1 5 10 15 Gly Gly 48 18 PRTArtificial Sequence HCV-Core derived peptides 48 Asp Val Lys Phe Pro GlyGly Gly Gln Ile Val Gly Gly Val Tyr Leu 1 5 10 15 Leu Pro 49 18 PRTArtificial Sequence HCV-Core derived peptides 49 Val Gly Gly Val Tyr LeuLeu Pro Arg Arg Gly Pro Arg Leu Gly Val 1 5 10 15 Arg Ala 50 18 PRTArtificial Sequence HCV-Core derived peptides 50 Gly Pro Arg Leu Gly ValArg Ala Thr Arg Lys Thr Ser Glu Arg Ser 1 5 10 15 Gln Pro 51 18 PRTArtificial Sequence HCV-Core derived peptides 51 Lys Thr Ser Glu Arg SerGln Pro Arg Gly Arg Arg Gln Pro Ile Pro 1 5 10 15 Lys Ala 52 18 PRTArtificial Sequence HCV-Core derived peptides 52 Arg Arg Gln Pro Ile ProLys Ala Arg Arg Pro Glu Gly Arg Thr Trp 1 5 10 15 Ala Gln 53 18 PRTArtificial Sequence HCV-Core derived peptides 53 Pro Glu Gly Arg Thr TrpAla Gln Pro Gly Tyr Pro Trp Pro Leu Tyr 1 5 10 15 Gly Asn 54 19 PRTArtificial Sequence HCV-Core derived peptides 54 Gln Tyr Pro Trp Pro LeuTyr Gly Asn Glu Gly Cys Gly Trp Ala Gly 1 5 10 15 Trp Leu Leu 55 17 PRTArtificial Sequence HCV-Core derived peptides 55 Cys Gly Trp Ala Gly TrpLeu Leu Ser Pro Arg Gly Ser Arg Pro Ser 1 5 10 15 Trp 56 25 PRTArtificial Sequence HCV-Core derived peptides 56 Trp Leu Leu Ser Pro ArgGly Ser Arg Pro Ser Trp Gly Pro Thr Asp 1 5 10 15 Pro Arg Arg Arg SerArg Asn Leu Gly 20 25 57 25 PRT Artificial Sequence HCV-Core derivedpeptides 57 Ser Trp Gly Pro Thr Asp Pro Arg Arg Arg Ser Arg Asn Leu GlyLys 1 5 10 15 Val Ile Asp Thr Leu Thr Cys Gly Phe 20 25 58 25 PRTArtificial Sequence HCV-Core derived peptides 58 Ser Arg Asn Leu Gly LysVal Ile Asp Thr Leu Thr Cys Gly Phe Ala 1 5 10 15 Asp Leu Met Gly TyrIle Pro Leu Val 20 25 59 25 PRT Artificial Sequence HCV-Core derivedpeptides 59 Leu Thr Cys Gly Phe Ala Asp Leu Met Gly Tyr Ile Pro Leu ValGly 1 5 10 15 Ala Pro Leu Gly Gly Ala Ala Arg Ala 20 25 60 25 PRTArtificial Sequence HCV-Core derived peptides 60 Tyr Ile Pro Leu Val GlyAla Pro Leu Gly Gly Ala Ala Arg Ala Leu 1 5 10 15 Ala His Gly Val ArgVal Leu Glu Asp 20 25 61 25 PRT Artificial Sequence HCV-Core derivedpeptides 61 Gly Ala Ala Arg Ala Leu Ala His Gly Val Arg Val Leu Glu AspGly 1 5 10 15 Val Asn Tyr Ala Thr Gly Asn Leu Pro 20 25 62 23 PRTArtificial Sequence HCV-Core derived peptides 62 Leu Glu Asp Gly Val AsnTyr Ala Thr Gly Asn Leu Pro Gly Cys Ser 1 5 10 15 Phe Ser Ile Phe LeuLeu Ala 20 63 23 PRT Artificial Sequence HCV-Core derived peptides 63Leu Pro Gly Cys Ser Phe Ser Ile Phe Leu Leu Ala Leu Leu Ser Cys 1 5 1015 Leu Thr Val Pro Ala Ser Ala 20

1. A method of simultaneously detecting at least one Hepatitis C Virus(HCV) antigen and at least one HCV antibody in a test sample comprisingthe steps of: a) contacting said test sample with: 1) at least one HCVviral antigen or portion thereof coated on a solid phase, for a time andunder conditions sufficient for the formation of antibody/antigencomplexes and 2) at least one antibody to HCV or portion thereof coatedon said solid phase, for a time and under conditions sufficient for theformation of antigen/antibody complexes; b) detecting saidantibody/antigen complexes, presence of said complexes indicatingpresence of HCV antigen in said test sample; and c) detecting saidantigen/antibody complexes, presence of said complexes indicatingpresence of HCV antibody in said test sample.
 2. The method of claim 1wherein said at least one HCV antigen coated on the solid phase isselected from the group consisting of core antigen, NS3, NS4, NS5, andportions thereof.
 3. The method of claim 2 wherein said at least oneantibody coated on said solid phase is a monoclonal antibody selectedfrom the group consisting of 13-959-270, 14-1269-281, 14-1287-252,14-153-234, 14-153-462, 14-1705-225, 14-1708-269, 14-1708-403,14-178-125, 14-188-104, 14-283-112, 14-635-225, 14-726-217, 14-886-216,14-947-104, 14-945-218, 107-35-54, 110-81-17, 13-975-157, 14-1350-210,C11-3, C11-7, C11-10, C11-14 and C11-15.
 4. The method of claim 3wherein said at least one antibody coated on the solid phase is notreactive with said at least one antigen coated on the solid phase. 5.The method of claim 1 wherein said at least one antibody is a HCVanti-core monoclonal antibody and said at least one antigen is arecombinant HCV core protein.
 6. The method of claim 5 wherein saidrecombinant core protein does not contain epitopes to which saidanti-core monoclonal antibody binds.
 7. The method of claim 1 whereinsaid solid phase is a microparticle.
 8. A method for simultaneouslydetecting the presence of at least one HCV antigen and at least one HCVantibody in a test sample comprising the steps of: a) contacting saidtest sample with: 1) at least one HCV viral antigen or portion thereofcoated on a solid phase, for a time and under conditions sufficient forthe formation of antibody/antigen complexes and 2) at least one HCVantibody or portion thereof coated on said solid phase, for a time andunder conditions sufficient for the formation of antigen/antibodycomplexes; b) adding a conjugate to the resulting antibody/antigencomplexes for a time and under conditions sufficient to allow saidconjugate to bind to the bound antibody in (a) (1), wherein saidconjugate comprises a second antibody attached to a chemiluminescentcompound capable of generating a detectable signal and simultaneouslyadding a second conjugate to the resulting antigen/antibody complexesfor a time and under conditions sufficient to allow said conjugate tobind to the bound antigen in (a) (2), wherein said conjugate comprises athird antibody attached to said chemiluminescent compound capable ofgenerating a detectable signal; and c) detecting said generated signal,presence of said signal indicating presence of at least one antigen insaid test sample selected from the group consisting of HCV antigen andHCV antibody.
 9. The method of claim 8 wherein said at least one HCVantigen coated on the solid phase is selected from the group consistingof core antigen, NS3, NS4, NS5, and portions thereof.
 10. The method ofclaim 9 wherein said at least one antibody coated on said solid phase isa monoclonal antibody selected from the group consisting of 13-959-270,14-1269-281, 14-1287-25, 14-153-234, 14-153-462, 14-1705-225,14-1708-269, 14-1708-403, 14-178-125, 14-188-104, 14-283-112,14-635-225, 14-726-217, 14-886-216, 14-947-104, 14-945-218, 13-975-157and 14-1350-210, 107-35-54, 110-81-17, C11-3, C11-7, C11-10, C11-14 andC11-15.
 11. The method of claim 10 wherein said at least one monoclonalantibody coated on the solid phase is not reactive with said at leastone antigen coated on the solid phase.
 12. A kit comprising: a) acontainer containing at least one HCV antigen coated on a solid phase;and b) a container containing at least one HCV antibody coated on asolid phase.
 13. A kit comprising: a container containing: 1) at leastone HCV antigen coated on a solid phase and 2) at least one HCVantibody, coated on said solid phase.
 14. The kit of claim 12 or claim13 further comprising at least one conjugate comprising asignal-generating compound attached.
 15. The kit of claim 14 whereinsaid signal-generating compound is acridinium.
 16. A method of detectingat least one HCV antigen in a test sample comprising the steps of: a)contacting said test sample with at least one HCV antibody coated on asolid phase, for a time and under conditions sufficient for theformation of antibody/antigen complexes; and b) detecting the presenceof antibody/antigen complexes, presence of said complexes indicatingpresence of said at least one HCV antigen in said test sample.
 17. Amethod of detecting at least one HCV antigen in a test sample comprisingthe steps of: a) contacting said test sample with at least one HCVantibody coated on a solid phase, for a time and under conditionssufficient for the formation of antibody/antigen complexes; b) adding aconjugate to the resulting antibody/antigen complexes for a time andunder conditions sufficient to allow said conjugate to bind to the boundat least one antibody, wherein said conjugate comprises a secondantibody attached to a chemiluminescent compound capable of generating adetectable signal; and c) detecting said signal generated by saidchemiluminescent compound, a signal generated by said chemiluminescentcompound indicating the presence of at least one HCV antigen in saidtest sample.
 18. A recombinant protein comprising an amino acid sequenceselected from the group consisting of SEQ ID NO:6, SEQ ID NO:8, SEQ IDNO:12, SEQ ID NO:16, and conservative amino acid substitutions thereof.19. A recombinant protein comprising an amino acid sequence encoded by anucleotide sequence selected from the group consisting of, for example,SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:11 and SEQ ID NO:15.
 20. A vector orconstruct comprising a nucleotide sequence selected from the groupconsisting of SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:11 and SEQ ID NO:15.21. A host cell comprising said vector or construct of claim
 20. 22. Animmunoassay which simultaneously detects at least one HCV antigen and atleast one HCV antibody in a test sample.